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G1S Cell Cycle Phase Marker Assay—User Manual

1. Cells that demonstrate BrdU incorporation indicative of active DNA replication consistently exhibit even or predominantly cytoplasmic distribution of the G1S CCPM Figure 5 13 confirming that the sensor is nuclear in G phase and is exported from the nucleus prior to DNA replication and entry into S phase Multiplexing the G1S CCPM with the Cell Proliferation Fluorescence Assay also facilitates the accurate discrimination of S phase and G phases of the cell cycle Figure 5 13 25 9003 97UM Rev A 2005 Chapter 5 25 450 400 Inuc Red NO oOo OO e S e N O e 150 100 0 8 N C ratio Green GFP BrdU Figure 5 13 Multiplex assay with G1S CCPM sensor and bromodeoxuyuridine BrdU incorporation imaged on IN Cell Analyzer 1000 A U 2 OS cell line exhibiting stable expression of the G1S CCPM sensor was incubated with BrdU for 1 hour and processed accordingly right bottom panel using the Cell Proliferation Fluorescence Assay see section 5 2 The graph left panel depicts analysis of the image bottom right and demonstrates that individual cells with a predominantly nuclear distribution of the G1S CCPM sensor high nuclear cytoplasmic ratio of green signal do not exhibit nuclear BrdU incorporation nuclear intensity red signal indicative of DNA replication activity and cells in S phase The graph also shows the ease of discrimination of S phase red box and G phase cells blue box using this assay on
2. Table 7 4 Results from a typical single assay performed using the suggested protocol Parameter Assay Data SD Assays Replicates Roscovitine ECs 32UM 96 Gi 17 8 1UM Nocodazole ECso 191nM 96 G 18 8 32nM Table 7 5 Summary results from assays performed by different operators on different occasions using the suggested protocol Assays were performed by three different operators six repeat assays per operator cells at P9 8 replicates per dose Data are mean SD SD shown are standard deviation of the assaus 8 Troubleshooting guide 8 1 Troubleshooting Problem Low cell number Low assay response Irregular cell morphology Low nuclear intensity Image is out of focus IN Cell Analyzer 3000 only Intensity variation across image field IN Cell Analyzer 3000 only 25 9003 97UM RevA 2005 Chapter 8 Possible cause Plating density too low high Cell cycle drugs block in mitosis Passage number too high Cell density too low or too high Incorrect selection of analysis parameters Incorrect assay incubation conditions Reagents were not stored properly or they are out of date Cells have been stressed during assay Loss of EGFP signal Over fixation and permeabilisation when multiplexing with Cell Proliferation Fluorescence Assay Harsh wash steps Nuclear stain concentration too low Nuclear stain incubation time too short Autofocus AF Offse
3. 5734 c 6096 6105 c 6240 6350 c 6471 c 6490 c 6617 c 6645 c 5199 957 1 1070 1398 1442 c 1641 2289 c 3970 4402 5134 6146 c o 1040 5678 5800 1245 1273 1423 1602 916 c 2985 c 333 677 1048 1054 1217 1229 1265 1298 1370 1403 1619 1667 1778 1952 2023 2514 2859 3116 3306 3594 3648 3930 4388 4749 4768 5447 5510 5610 6131 6388 6434 6524 88 c 282 386 1739 c 1938 2632 c 2641 3235 4003 4068 c 4249 4613 c 4626 5161 5165 c 5482 5945 c 5946 6042 c 6044 6130 62 9 3376 3448 802 4631 5128 6374 1181 2263 2309 2475 3129 3140 5625 561 1075 2381 498 715 973 1901 2040 4540 5436 5658 940 3630 175 4618 21 54 703 788 967 1111 1221 1280 2905 5280 5825 5858 5847 1251 2025 2875 4189 25 9003 97UM Rev A 2005 Chapter 11 37 Enzyme t of cuts Positions Bbel Bbr7l Bbsl Bbvl Bccl BceAl Bcefl Bcgl BciVI Bfal BfrBI Bgll Bglll Bme1580l BmgBl Bmrl Bmtl Bpll Bpml Bpu10 Bpuel BsaAl BsaBl BsaHl Bsal BsaJl BsaWl Bsax BscAl BseM I BseRl BseY Bsgl BsiEl BsiHKAI Bsll 4 50 18 16 16 tS p pm P N WO NN WW NN DMB pL OD fno NO UT 25 50 9 1 5827 588 2282 c 583 2287 c 45 c 92 115 c 357 398 c 553 1390 c 1496 1780 1787 1813 c 1816 c 1993 2501 c 2708 2776 3247 3771 c 3897 3939 3955 c 4048 c 4460 4755 c 5366 c 5757 6060 c 6266 c 6269 c 6359 687 c 715 1293 1545 1815 2152 c 2172 c 2931 c 2963 3502 3702 c 4044 4130 c 4539 c 4546 5343 c
4. Protective clothing such as laboratory overalls safety glasses and gloves should be worn whenever genetically modified materials are handled 3 Avoid actions that could lead to the ingestion of these materials and NO smoking drinking or eating should be allowed in areas where genetically modified materials are used 4 Any spills of genetically modified material should be cleaned immediately with a suitable disinfectant 5 Hands should be washed after using genetically modified 25 9003 97UM RevA 2005 Chapter 4 materials 6 Care should be taken to ensure that the cells are NOT warmed if they are NOT being used immediately To maintain viability DO NOT centrifuge the cells upon thawing 7 Most countries have legislation governing the handling use storage disposal and transportation of genetically modified materials The instructions set out above complement Local Regulations or Codes of Practice and users of these products MUST make themselves aware of and observe the Local Regulations or Codes of Practice which relate to such matters For further information refer to the material safety data sheet s and or safety statement s contained within this product 4 2 Storage The pCORON1002 EGFP C1 PSLD expression vector NIF2052 should be stored at 15 C to 30 C U 2 OS cells exhibiting stable expression of the EGFP PSLD fusion protein from the above vector NIF2053 should be stored at 196 C in liquid nitroge
5. Vector use details The plasmid vector pCORON1002 EGFP C1 PSLD Figure 3 1 can be used to express transiently or stably the G1S CCPM sensor in a cell line of choice 6 1 General guidelines for vector use U 2 OS cells were used to generate stable cell lines with the vector according to method summarized in section 3 3 2 The user must be aware that the genotype of the cell line will govern how the sensor performs Changes in the level of a single component of the cell cycle machinery can disrupt the complex interplay between CDKs inhibitors and cyclins throughout the cell cycle 6 2 Transfection with pCORON1002 EGFP C1 PSLD Transfection protocols must be optimised for the cell type of choice Both transfection reagent and cell type will affect efficiency of transfection The following standard protocol in used for adherent cells and may serve as a useful guideline for establishing an appropriate protocol For more information refer to manufacturer s guidelines for the chosen transfection reagent 6 2 1 FUGENE 6 Transfection Reagent protocol Day 1 Seed cells so that the density will be 50 80 confluent the next day Day 2 1 Add serum free media to an empty tube 2 Add FUGENE 6 Transfection Reagent directly into this medium dropwise Mix by gentle pipetting 3 Add the FUGENE 6 Transfection Reagent medium mix to the tube containing the DNA Mix by gentle pipetting 4 Incubate for a minimum of 15 min at room te
6. 1048 1054 1189 1199 1217 1229 1265 1298 1518 15 0 1405 1418 1462 1519 1619 1667 1687 1707 1778 1819 1952 2025 2118 2551 2589 2514 2645 2665 2699 2845 2859 28 5 2954 5096 5116 5246 5506 5547 5419 5542 5571 5577 5586 5594 5617 5629 5655 5648 5684 5690 3732 3749 3757 3784 5809 3893 5902 3906 3930 3968 4044 4061 4080 4143 4187 4197 4214 4297 4324 4328 4365 4388 4404 4549 4672 4676 4716 4749 4768 4822 5409 5447 5510 5520 5610 5676 5685 5689 5715 5756 5768 6131 6160 6203 6214 6279 6358 6383 6388 6434 6524 6622 6648 6666 6677 6693 112 147 1848 1866 2371 3288 3590 4487 4638 4873 5299 5839 6005 6414 95 276 580589 1746 1894 1932 2655 26592675 32295997 4075 4156 4165 4243 4620 5119 5155 5172 5430 5476 5494 5835 5940 5952 6030 6038 6049 6124 6702 2591 5222 5914 5955 599 2949 2993 3667 3851 4717 6586 421 436 1187 1316 1705 2387 3730 3904 4295 4322 4547 5407 2387 3730 2013 c 2126 2654 c 4168 c 4378 c 5001 c 90 c 1493 c 1737 c 1850 c 3308 3479 c 3500 c 3510 c 3521 c 4353 5658 6486 6632 S 763 1070 1398 1442 c 1641 1689 1929 1978lc 2047 2129 2289 c 3970 4402 5134 5731 6146 c 11 103 318 348 423 441 1272 1926 2380 2381 2640 3826 3986 4726 4761 5262 5613 6309 12292025 944 715 2040 4820 1255 962 1252 1295 1509 1616 1651 1760 2570 2396 2657 2912 3674 5958 4094 4118 4154 4316 4507 4606 5147 6591 6696 1754 3287 4164 624 1243 1477 1507 1702 1897 1942 2089 2403 3241 3374 3446 3537 3694 4039 4225 4256 4282 4771 4855 4960
7. 10mM Tris 1mM EDTA pH8 0 NIF2052 e Clonal U 2 OS cells exhibiting stable expression of the G1S CCPM 2 vials each containing 1x106 cells Supplied in 1ml of 1096 v v DMSO and 90 v v FBS NIF2053 e User manual 5 2 GFP expression vector pCORON1002 EGFP C1 PSLD The supplied vector DCORON1002 EGFP C1 PSLD is 6 704kb and contains a bacterial ampicillin resistance gene and a mammalian neomucin resistance gene The sequence of the construct is available on request A detailed restriction map is available in Chapter 11 BglII 6700 Eco 47III 57 Ubiquitin C promotor Amp R HindIII 1215 Eco RV 1235 pCORON1002 EGFP C1 PSLD EGFP 6704 bp Bsr GI 1954 Nhe I 1977 PA Bam HI 4618 gt P PSLD Synthetic poly A Sal I 2376 Neo f Xma I 2381 HindIII 3646 Sma I 2383 SV40 minimum origin of replication Not I 2387 SV40 enhancer early promoter SV40 late polyA f1 ori Figure 3 1 Vector map of pCORON1002 EGFP C1 PSLD expression vector 3 3 Clonal U 2 OS cells exhibiting stable expression of the G1S CCPM 3 3 1 U 2 OS parental cell line U 2 OS ATCC HTB 96 are human osteosarchoma cells derived from the thighbone of a 15 year old Caucasian female 7 8 9 U 2 OS cells were chosen to generate a cell line exhibiting stable expression of the G1S CCPM sensor since they are p53 and Rb competent 10 However U 2 OS cells contain a disrupted CDKN2A locus resulting in a lack of expression of
8. 3452 3378 3450 4229 4775 1525 4761 133 c 190 c 572 3002 3010 c 4489 c 5809 6312 c 1251 c 1569 c 2985 c 5172 5982 c 55 6 5448 15 2040 867 2510 3074 501 718 976 1904 2043 4343 5439 5661 1288 1413 1525 1600 1654 2106 2334 3342 3397 3414 4209 6527 6540 6661 718 2043 4823 3877 2676 5431 5506 5950 2567 c 811 1030 1223 1674 1956 3282 4130 4643 5319 4540 12512025 Ie 2516 1255 2013 c 4168 c 4378 c 123 444 498 715 880 937 973 1417 1818 1901 2040 2664 2952 4340 4820 5436 5658 5675 5754 3319 13 105 320 350 425 443 1274 1290 1415 1527 1602 1656 1928 2108 2336 2382 2383 2642 3344 3399 3416 3828 3988 4211 4728 4763 5264 5615 6311 6529 6542 6663 7 29 314 361 501 822 824 1571 1889 2211 2716 2740 2760 3136 3223 3888 4189 4221 4622 4702 4805 4807 4907 5239 5 52 6062 6643 559 1343 c 1621 1636 1735 2067 2226 2473 c 3165 c 3205 3387 3459 3782 c 4037 c 4123 4187 4253 c 4462 4646 c 4740 5099 c 5348 5539 c 6591 c 488 2071 2 25 58 5 5554 6232 6423 5505 25560909 5525 359 498 715 c 1137 1372 c 1567 c 1874 c 2040 c 2378 0c 2912 c 4252 c 5728 6014 c 6497 2585 651 1040 3678 4215 5175 6045 6520 6582 1165 55 8 5450 4229 205 301 411 700 839 c 993 1259 1261 c 1592 1616 1745 2095 c 2134 c 2163 c 2946 4001 c 4779 c 4788 c 25 9003 97UM RevA 2005 Chapter 11 43 Enzyme ofcuts Positions SspD5l Cont d Sspl Sth132l Stsl Stul StyD4l Styl Tail Tagl Taqli Tatl
9. 9003 97UM RevA 2005 Chapter 10 36 Code 25 8010 50 25 9001 89 25 8007 26 25 8007 27 25 8008 82 25 8010 17 25 8010 21 20 8010 38 25 8010 42 25 8010 46 25 8010 11 28 4026 98 65 0050 1 63 0055 60 25 8010 26 25 8098 11 11 Appendix 11 1 Restriction map of pCORON1002 EGFP C1 PSLD The following enzymes do not cut the vector Afllll Agel Apal Ascl AsiSI BbvCI Bcll Blpl BsiWI BspEI BstEll BstZ171 Bsu36l EcoRI Fsel FspAI Mlul Ndel Pacl Pcil PfIMI Pmel Pmll PShAI PspOMI Psrl Sbfl Scil SgrAl SnaBl Srfl Sse232I Sse86471 Swal Xbal Xcml Xhol Enzyme ofcuts Positions Aarl Aatil Accobl Accl Acelll Acil Acll Acul Afel ATI Ahdl Alel Alol Alul AlwNI ApaBbl ApaLl Apol Asel Aval Avall Avril Bael BamHI Banl Banll Oo e NN Be N A e N rn WO N eL e e N O C e 0 AA N m WN 894 848 4881 1221 3280 2501 1251 c 1284 c 1605 c 1788 1938 c 3102 c 4754 c 5496 c 7 29 31 c 101 121 c 126 227 c 268 c 287 c 363 c 407 c 439 447 478 501 552 c 591 c 633 c 669 c 768 c 774 c 786 c 901 c 1176 1463 1504 1571 1610 1748 1861 1921 1924 1969 c 1984 c 2386 c 2390 2667 27281c 2742 c 2745 c 2773 2800 3178lc 3204 c 3217 3225 c 3293 c 3478 3490 3499 3511 3521 3532 3578 3733 3796 3890 c 3954 c 4055 c 4058 c 4298 4338 c 4343 4393 c 4409 4435 4491 c 4550 4622 4660 4686 4696 4735 4909 c 4956 5055 c 5164 c 5241 c 5285 5406 c 5452 5643 c
10. Taul Tfil Tsel Tsp45l Tsp509 TspDT TSpGWI TSpRI Tth111l Tth111ll Unbl UthSI VpaK11AI mal mnl Zral e 65 10 32 16 22 50 50 les 19 10 15 N enre O e 5072 c 5108 5313 c 5730 5957 5154 4995 4 c 23 c 35 111 132 175 c 311 c 341 c 399 c 416 c 434 c 568 583 c 661 c 67 7 c 760 c 814 858 927 c 1068 c 1265 c 1414 1477 1519 1577 1828 1840 1919 c 2374 c 2388 2633 c 2673 273 7 c 2806 2858 2859 c 3484 3505 3834 3904 c 3946 c 3979 c 4237 4376 4447 4483 c 4685 4692 4702 4704 c 4734 4754 c 4887 c 5197 5255 c 5258 5621 5780 5788lc 6025 6302 c 6346 c 6355 c 6463 6554 c 1271 c 1637 c 2203 c 3480 c 4149 4174 4717 c 5362 5649 5350 657 2118 3629 11 103 318 348 423 441 1272 1288 1413 1525 1600 1654 1926 2106 2334 2380 2381 2640 3342 3397 3414 3826 3986 4209 4726 4761 5262 5613 6309 6527 6540 6661 481 644 940 1243 3241 3537 3630 4256 67 726 848 1312 1525 1696 2838 2948 2991 3003 3943 4130 4881 5201 5574 5990 96112351 1294 1588 1615 1630 1 59 2377 2595 26560 2911 3673 3937 4093 4117 4153 4315 4506 4605 5146 6590 6695 3044 c 3861 4529 5268 c 5453 1672 1954 4641 5317 124 441 449 555 636 777 1465 1926 1972 1987 2389 2392 2731 2745 5580 5 55 3798 5895 4058 4061 4500 4396 4437 4552 4662 5058 5287 5409 6493 6648 262 958 2280 5652 4309 4443 4602 58 80 128 345 411 541 1403 1484 1768 1775 1826 1829 1981 2514 2696 2764 3235 3784 3885 3927 3968 4061
11. a non confocal imaging system The ability to correlate multiparametric data obtained on the cell cycle using the G1S CCPM with replication activity per cell using BrdU and DNA complement per cell using the DNA marker is an additional and very compelling reason to carry out this multiplexed assay Figure 5 14 The process can be used to derive temporal information from end point assays and indicate relatively complex drug effects 25 9003 97UM RevA 2005 Chapter 5 26 9 9 O Oo LH 56 OControl 24h o Boe p me ee WV o 9 o O Compound A 24h 96 oq 99 5 4 Replicating cells T T A O co Un N Log BrdU Cy5 Fluorescence RB 4 2 e o e o e e 2n 4n an 3 8 l 4 6 4 85 5 1 5 35 5 6 Log DNA Hoechst Fluorescence 5 8 4 o 56 OControl48h O Compound A 48h 5 4 Replicating cells a i a Oo co Cn Ny Log BrdU Cy5 Fluorescence E T 4 2 4n an 3 8 T T T 4 6 4 85 5 1 5 35 5 6 Log DNA Hoechst Fluorescence 5 5 5 1 G1S CCPM fixed cell assay multiplexed with Cell Proliferation Fluorescence Assay to produce Roscovitine dose response Example images demonstrating multiplexing of G1S CCPM with the Cell Proliferation Fluorescence Assay imaged on the IN Cell Analyzer 3000 are shown in Figure 5 15 LOADER 25 9003 97UM Rev A 2005 Chapter 5 27 Figure 5 14 Multiplex assay showing effect of a compound that causes endoreduplication after 2
12. all cells are in contact with the solution 5 Immediately aspirate the Trypsin EDTA from the cells and discard 6 Incubate at 37 C 5 CO 95 humidity for 2 3minutes for the cells to round up loosen and detach Check on an inverted microscope 7 When the cells are loose tap the flask gently to dislodge the cells Add 6ml of Assay medium and resuspend the cells by gentle agitation with a 10ml pipette until all clumps have dispersed 8 Count the cells using either a CASY1 Cell Counter and Analyzer System Model TT or a hemocytometer 9 Using fresh Assay medium adjust the cell density so that it will deliver the desired number of cells to each well For example to plate 5000 cells per well in 100ul of suspension the suspension is adjusted to 5 x 104 cells per ml 10 Incubate the plated cells for 24 hours at 37 C 5 CO gt 95 humidity before starting the assay 5 1 6 Cell cryopreservation procedure 1 Harvest the cells as described in section 5 1 5 and prepare a cell suspension containing 1 x 106 cells per ml 2 Pellet the cells at approximately 1000g for 5 minutes Aspirate the medium from the cells 3 Resuspend the cells in cryopreservation medium until no clumps remain and transfer into cryo vials Each vial should contain 1 x 106 cells in 1ml of cryopreservation medium 4 Transfer the vials to a cryo freezing device and freeze at 80 C for 16 24 hours 5 Transfer the vials to the vapor phase in a liquid nit
13. analysed further Clones were selected on the basis of growth rate expression of the sensor level homogeneity and temporal consistency and cell morphology Clones were characterised biologically with sIRNA and cell cycle inhibitors Clones were validated by time lapse microscopy and through comparison with other methods of assessing the cell cycle distribution for a population U 2 OS cells exhibiting clonal stable expression of the G1S CCPM that met specification were processed further and one of these lines 22 7 is supplied The cells are mycoplasma negative 3 4 Materials and equipment The following materials and equipment are required but not provided see also chapter 5 e Microplates For analysis using the IN Cell Analyzer 5000 Packard Black 96 Well ViewPlates Packard Cat 6005182 are recommended For assays in 384 well format please contact your local GE Healthcare representative for recommendations For analysis using the IN Cell Analyzer 1000 Greiner uClear 96 well microplates Cat 655090 are recommended e A CASY 1 Cell Counter and Analyzer System Model TT Sch rfe System GmbH is recommended to ensure accurate cell counting prior to seeding Alternatively a hemocytometer may be used e Environmentally controlled incubator 596 CO 95 relative humidity 37 C e Sub cellular imager cellular microscope e g IN Cell Analysis System e Controlled freezing rate device providing a controlled freezi
14. and a C terminal location control element Figure 1 2 Helicase B displays strong ATPase activity in the presence of single stranded DNA and exhibits 5 3 DNA helicase activity dependent upon Walker A and B motifs Helicase B associates phusically and functionally with DNA polymerase a primase and mutational studies have shown that enzyme activity is essential for G S transition of the cell cycle Gu et al 3 have demonstrated that the protein is localized at nuclear foci induced by DNA damage and have indicated that helicase B operates primarily in G4 to process endogenous DNA damage prior to the G S transition Consistent with the proposed action of helicase B the protein resides in the nucleus of G phase cells but is predominantly cytoplasmic in S and G phase cells Gu et al 3 have demonstrated that a 131 amino acid C terminal phosphorylation dependent subcellular localisation control domain PSLD of helicase B is portable and contains active targeting signals that are independent of protein context The PSLD contains a nuclear localisation sequence NLS a rev like nuclear export sequence NES and seven putative cyclin dependent kinase Cdk phosphorylation sites Figure 1 2 Phosphopeptide mapping demonstrated that of the seven putative phosphorylation sites serine 967 is the primary site of phosphorylation by Cdks in vivo and in vitro In addition the phosphorylation of S967 is coincident with G4 S transition and is mai
15. cells and discard 4 Add Trypsin EDTA 2ml for T 75 flasks and 5ml for T 175 flasks ensuring that all cells are in contact with the solution 5 Immediately aspirate the Trypsin EDTA from the cells and discard 25 9003 97UM RevA 2005 Chapter 5 15 6 Incubate at 37 C 5 CO 95 humidity for 2 3 minutes for the cells to round up loosen and detach Check on an inverted microscope 7 When the cells are loose tap the flask gently to dislodge the cells Add 10ml of Growth medium and resuspend the cells by gentle agitation with a 10ml pipette until all clumps have dispersed 8 Aspirate the cell suspension and dispense 1 2ml of the cell suspension into a new culture vessel containing sufficient fresh Growth medium 5 1 5 Cell seeding procedure This procedure has been developed for cells grown in a standard T 175 flask and seeded into microplates All reagents used for seeding cells should be maintained at 37 C If the cells are near confluence prior to seeding to ensure the cell population is in the log phase of growth they should be sub cultured at a ratio of 1 2 into two T 175 flasks The actively growing cells will be ready for seeding the following day 1 Aspirate the medium from the cells and discard 2 Wash the cells with 10ml PBS Take care not to damage the cell layer while washing but ensure that the entire cell surface is washed 3 Aspirate the PBS from the cells and discard 4 Add 5ml Trypsin EDTA ensuring that
16. considerations 2 1 Licensing considerations 2 2 Legal 3 Product contents 5 1 Components summary 5 2 GFP expression vector pCORON1002 EGFP C1 PSLD 3 3 Clonal U 2 OS cells exhibiting stable expression of the G1S CCPM 5 5 1 U 2 OS parental cell line 5 3 2 Production of clonal U 2 OS cells exhibiting stable expression of the G1S CCPM 5 4 Materials and equipment 5 5 Software requirements 5 5 1 IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module 3 5 2 IN Cell Analyzer 1000 G1S Cell Cycle Trafficking Analysis Module 3 5 3 Confocal or epifluorescence microscopes 3 5 4 IN Cell Image Converter 123 4 Safety warnings handling and precautions 4 1 Safety warning 4 2 Storage 4 3 Handling 4 5 1 Vector 4 3 2 Cells 5 Cell assay design 5 1 Culture and maintenance of U 2 OS cell line exhibiting stable expression of the G1S CCPM 5 1 1 Tissue culture media and reagents required 5 1 2 Reagent preparation 5 1 3 Cell thawing procedure 5 1 4 Cell sub culturing procedure 5 1 5 Cell seeding procedure 5 1 6 Cell cryopreservation procedure 25 9003 97UM RevA 2005 Pagefinder 8 o xx oa aa OY 10 10 10 11 11 11 11 11 11 11 12 12 12 12 12 13 15 15 15 15 13 14 14 14 14 15 15 16 16 5 1 7 Growth characteristics 5 2 Assay set up 5 2 1 General Assay Set up 5 2 2 G1S CCPM fixed cell assay procedure 5 2 2 1 G1S cell cycle synchronization
17. of EGFP via a short flexible amino acid linker region The sensor is expressed from the human ubiquitin C promoter UbC 25 9003 97UM Rev A 2005 Chapter 1 1 2 2 Ubiquitin C promoter The human ubiquitin C promoter is highly active in many mammalian tissues and has been reported to provide the most reliable expression in a range of cell types 4 5 Expression levels generated by the ubiquitin C promoter are around one half to one third the levels obtained with the CMV I E promoter dependent upon the cell type but the ubiquitin C promoter has been shown to be less prone to tissue specific or temporal inactivation 1 2 3 The G1S CCPM sensor The G1S CCPM sensor is a fusion of the human helicase B PSLD region to the C terminus of EGFP Figure 1 2 The sensor contains an amino acid linker region that permits flexibility between the two component peptides facilitating access of the cell cycle machinery to the PSLD The linker also increases the overall size of the sensor above 40kDa precluding passive diffusion across the nuclear membrane 6 Therefore the sensor must be actively transported from the nucleus resulting in a precise indication of cell cycle progress The ubiquitin C promoter is included in the plasmid vector construct that has been used in the production of clonal U 2 OS cells exhibiting stable expression of the G1S CCPM sensor since low homogeneous and consistent levels of ectopic expression are desirable in o
18. response curve e SM 300 16 hours for U 2 OS cells exhibiting stable expression of G1S CCPM EC values of 34nM Nocodazole 96 of cells in G and 50nM Nocodazole 96 of cells in M phase were 100 calculated from the dose response 1 SET curve Mean SD n 8 replicates per dose R2 0 63 and 0 71 90 85 80 75 70 65 60 55 respectively N e e Jequunu 92 Nocodazole M 5 5 5 Multiplexing the G1S CCPM with a marker of DNA replication activity This confirms sub cellular relocation of the sensor prior to S phase and can increase cell cycle discrimination and information content of an assay The export of the G1S CCPM from the nucleus is a progressive event throughout the cell cycle and provides an indication of the cell cycle phase of individual cells see for example Figure 1 4 and Figure 1 5 High levels of Cdk2 cuclin E complex activity in late G4 phase result in dramatic changes in the sub cellular distribution of the G1S CCPM sensor providing an accurate means to discriminate G phase and S phase However subtle changes in the subcellular distribution of the G1S CCPM sensor can make discrimination of S phase and G phase less precise on non confocal imaging devices since the dynamic range can be markedly reduced Multiplexing the G1S CCPM with a BrdU based incorporation assay such as the Cell Proliferation Fluorescence Assay see section 5 2 for method can provide improved discrimination of the cell cycle
19. through the use of phenol red free media and quality media components High quality plates will provide optical clarity Day 4 Ensure all solutions are pre warmed to 37 C prior to use unless otherwise stated 1 Remove cell cycle synchronization plate from incubator 2 Add 50ul of 8uM Hoechst in Assay medium to all wells providing a 2uM final Hoechst concentration 3 Return plate to incubator and incubate at 37 C 5 CO gt 95 humidity for 20 minutes 4 Remove plate from incubator and image 5 2 4 G1S CCPM live cell kinetic procedure Proceed as Days 1 and 2 for the assay procedure described in section 5 2 2 On day 3 add drugs according to methods in sections 5 2 2 1 and 5 2 2 2 and image during the incubation period 5 3 Imaging on IN Cell Analysis Systems The CCPM assays have been designed as part of the IN Cell Analysis System and optimal results are obtained if the assay is performed on an IN Cell Analyzer 3000 or an IN Cell Analyzer 1000 instrument Further advice on performing the G1S CCPM assay as part of these systems are included in the IN Cell Analyzer 1000 User manual the IN Cell Analyzer 3000 User manual and the IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module User manual 5 3 1 IN Cell Analyzer 3000 When performing the assay on an IN Cell Analyzer 3000 in 96 well format it is recommended to use a Packard View 96 well microplate If planning to perform the assay in 384 format please contact
20. using Roscovitine 5 2 2 2 G2M cell cycle synchronization using Nocodazole 5 2 3 G1S CCPM live cell end point assay procedure 5 2 4 G1S CCPM live cell kinetic procedure 5 3 Imaging on IN Cell Analysis Systems 5 3 1 IN Cell Analyzer 3000 5 3 1 1 Fixed cell imaging of G1S CCPM on the IN Cell Analyzer 3000 5 3 1 2 Live cell end point imaging of G1S CCPM on the IN Cell Analyzer 3000 5 3 1 3 Live cell kinetic imaging of G1S CCPM on IN Cell Analyzer 3000 5 3 1 4 Analysis using the IN Cell Analyzer 3000 5 3 2 IN Cell Analyzer 1000 5 4 Performing cell cycle phase marker assays on epifluorescence microscopes 5 5 G1S CCPM sensor and assay characterization 5 5 1 G1S CCPM fixed cell assay Roscovitine dose response 5 5 2 G1S CCPM fixed cell assay Nocodazole dose response 5 5 3 G1S CCPM live cell end point assay Roscovitine dose response 5 5 4 G1S CCPM live cell end point assay Nocodazole dose response 5 5 5 Multiplexing the G1S CCPM with a marker of DNA replication activity 5 5 5 1 G1S CCPM fixed cell assay multiplexed with Cell Proliferation Fluorescence Assay to produce Roscovitine dose response 5 5 5 2 G1S CCPM fixed cell assay multiplexed with Cell Proliferation Fluorescence Assay to produce Nocodazole dose response 5 5 5 3 Imaging the G1S CCPM assay multiplexed with Cell Proliferation Fluorescence Assay on the IN Cell Analyzer 1000 5 5 6 Characterisation of G1S CCPM with inhibitors of the cell cycle 5 5 7
21. 0 2755 2 42 27 64 2790 2798 32355 3818 3826 3890 3927 4193 4223 4225 4453 4706 4809 4909 5241 5578 5671 6064 6173 6347 6447 6514 20 1992 c 2024 3987 c 4017 c 4019 5712 c 5786 c 5818 922 39 560 170 516 559 022 624 820 960 1072 1552 15 5 1889 1987 2694 2718 2731 2740 2762 2788 2796 3233 3816 5824 5888 5925 4191 4221 4223 4451 4704 4807 4907 5239 5576 5669 6062 6171 6345 6445 6512 25182550 1215 3646 139 196 262 564 958 2280 2994 3016 3652 4309 4443 4495 4602 5801 6318 2530 12 105 319 349 424 442 890 971 1057 1273 1336 1396 1927 2382 2641 2842 3729 3806 3828 3856 3987 4077 4144 4325 4728 4762 5263 5505 5615 5682 5716 6120 6310 6336 6483 205 301 411 700 840 c 993 1259 1262 c 1592 1616 1745 2096 c 2135 c 2164 c 2946 4002 c 4780 c 4789 c 5073 c 5108 5314 c 5730 5957 69 2308 2627 2889 3261 3289 3591 3660 3791 4135 4488 4655 4780 5302 5422 5433 5879 6014 6148 6501 6579 91 114 220 561 568 651 841 955 1427 1742 1916 2026 2212 2299 2364 3172 3600 3672 3842 4158 4167 4250 4306 4350 4440 4473 4542 4826 4856 4961 5710 5969 6043 6126 6224 6322 6456 292 804 1270 1313 1607 1736 2378 2530 2943 2998 3015 4633 5130 5888 6376 345 837 921 1311 1557 1818 3914 3942 4236 4251 4528 5535 5798 6592 67 c 107 162 c 262 668 1509 c 1616 c 2690 2830 3294 3952 c 4469 4479 5076 5435 c 5688 6143 c 6453 c 25 9003 97UM Rev A 2005 Chapter 11 41 Enzyme ofcuts Positions HpyCHAlll HpyCHa4IV HpyCHA4V HpyF10VI Kasl Kpnl Lon
22. 1 5th of the original image generated by the IN Cell Analyzer Figure 5 19 shows a Roscovitine dose response curve for the G1S CCPM assay 1000 25 9003 97UM Rev A 2005 Chapter 5 28 when multiplexed with Cell Proliferation Fluorescence Assay Cells were incubated in the presence of Roscovitine for 24 hours prior to BrdU incorporation fixation antibody labelling and imaging on the IN Cell Analyzer 1000 The images obtained from the IN Cell Analyzer 1000 were converted using the IN Cell Image Converter 123 and analysis was performed using the G1S Cell Cycle Trafficking Analysis Module Based on the percentage of cells in G phase of the cell cycle an EC of 32uM was determined This value compares directly with that obtained on the IN Cell Analyzer 3000 see section 5 5 5 1 80 Figure 5 19 Roscovitine dose response curve 24 hours for fixed cell G1S CCPM assay multiplexed with Cell Proliferation Fluorescence Assay imaged on the IN Cell Analyzer 1000 converted and analysed using the G1S Cell Cycle Trafficking Analysis Module An EC of 32uM Roscovitine of cells in G was calculated from the dose response 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 curve Mean SD n 8 replicates Roscovitine M per dose R 0 74 70 60 50 40 Jequunu jeD 30 20 G1 Cell number Figure 5 20 shows a Nocodazole dose response curve for the G1S CCPM assay when multiplexed with the Cell Proliferation Fluorescenc
23. 4 hours and arrest in G phase at 4n and 8n after 48 hours U 2 OS cells exhibiting stable expression of the G1S CCPM sensor were grown for 24 hours top panel or 48 hours bottom panel in the presence red or absence of compound A blue pulse incubated with BrdU for 1 hour fixed and processed accordingly Graph shows an object plot of individual cells imaged on the IN Cell Analyzer 1000 and analysed twice with the Morphology Analysis Module GE Healthcare The DNA replication activity BrdU incorporation per nucleus is indicated on the y axis and is a measure of the log of the integrated total red fluorescence due to Cy5 labelled anti BrdU per nucleus replicating cells are shown above the dotted line The DNA content per nucleus is indicated on the x axis and is a measure of the integrated blue fluorescence due to Hoechst 33342 per nucleus nuclear DNA complement has been indicated at 2n 4n and 8n The cell cycle phase of each cell is indicated by the size of each point and is a measure of the nuclear cytoplasmic ratio of G1S CCPM sensor Larger dots are G phase cells smaller dots are S phase and G phase cells Cells exhibiting DNA replication from 2n to 4n are clearly visible in control populations at 24 and 48 hours and cell cycle distribution is normal Treated cells exhibit reduplication from 4n to 8n at 24 hours and many cells seem to have arrested in G phase at 4n or 8n after 48 hours Figure 5 15 Fixed
24. 4 hours incubation with Roscovitine 250uM and C 16 hours incubation with Nocodazole 10M Images shown are a 1 5th section of the original image 5 4 Performing cell cycle phase marker assays on epifluorescence microscopes For speed and quality of the images obtained GE Healthcare recommends performing the G1S CCPM assay on either the IN Cell Analyzer 3000 or the IN Cell Analyzer 1000 However it is possible to adapt the assay to be read and analysed on alternative imaging platforms Laboratory grade inverted epifluorescence microscopes such as the Nikon Diaphot or Eclipse models or the Zeiss Axiovert model are suitable for image acquisition A high quality objective Plan Fluor 40x 1 3NA or similar and epifluoresence filter sets compatible with GFP and the desired nuclear dye if used will be required A motorized stage with multi well plate holder and a heated stage enclosure are also recommended for assays performed on epifluorescence microscopes and a suitable software package will be required for image analusis For example Figure 5 7 shows time lapse images and Figure 5 8 the analysis from a live cell kinetic assay where asynchronous U 2 OS cells exhibiting stable expression of the G1S CCPM were followed over 24 hours on an Axiovert 100 microscope Carl Zeiss Welwyn Garden City UK The microscope was fitted with an environmental chamber capable of maintaining the stage at 37 C 1 C and 5 CO Solent Scientific Por
25. 4448 4768 5379 5745 6073 6279 6282 6347 193 391 733 841 846 1427 1916 2075 2102 2768 3944 4250 4751 5327 5538 1147 1169 1181 21652265 2309 2524 2529 24 5 2559 5129 5140 3166 5584 3456 5548 5367 5622 5928 1375 c 1494 2263 2537 c 3883 4301 c 4561 c 5530 5833 5910 c 269 c 329 c 474 905 c 1996 c 2030 5019 c 5361 212 742 806 1160 1458 2039 2456 3953 5381 5408 5755 5860 6009 6280 6293 3942 361 c 1728 2273 4259 c 6065 c 6097 6104 c 122 443 497 714 879 936 972 1416 1817 1900 2039 2663 2951 4339 4819 5435 5657 5674 5753 2582 497 714 972 1900 2039 4339 5435 5657 2581 5200 846 4879 25 9003 97UM RevA 2005 Chapter 11 44 25 9003 97UM RevA 2005 45 25 9003 97UM RevA 2005 Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE imagination at WOrk re 44 01800 515 313 Fax 44 01800 616 927 http www amershambiosciences com
26. 5 5 5 0 4 5 4 0 3 5 3 0 Roscovitine M 25 9003 97UM Rev A 2005 Chapter 5 24 5 5 4 G1S CCPM live cell end point assay Nocodazole dose response Figure 5 12 shows a Nocodazole dose response curve for a live cell G1S CCPM assay Cells were incubated in the presence of Nocodazole for 16 hours prior to addition of Hoechst nuclear marker and live cell end point imaging on the IN Cell Analyzer 3000 Analysis was performed using the G1S Cell Cycle Trafficking Analysis Module In contrast to the results of fixed cell assays the live cell protocol resulted in a decrease in the percentage of cells with a G phenotype with increasing concentration of Nocadazole section 5 5 2 Unlike fixed cell assays live cell assays do not include wash steps resulting in the detection of greater numbers of M phase and other poorly adhered cells and a more accurate representation of cell cycle arrest An EC of 50nM was determined through analysis of cells with an M phase phenotype indicating that the live cell assay produces a more sensitive method for the detection of the action of Nocodazole than that obtained with the fixed cell method section 5 5 2 However the differentiation of M phase cells from cells that have rounded due to necrosis may present a problem with this assay In such cases addition of propidium iodide 10uM prior to imaging will stain dead cells E G2 id Figure 5 12 Live cell assay Cell number Nocodazole dose
27. 5353 5389 5467 5477 5968 6688 24 c 36 133 400 c 584 c 662 c 761 c 1578 2674 2738 0c 2807 3485 3506 3947 c 4484 c 4693 4703 25 9003 97UM RevA 2005 Chapter 11 40 Enzyme ofcuts Positions Fmul Fnu4HI Fokl Fspl Gdill Hael Haell Haelll HaelV Hgal Hhal Hin4l HinP1l Hincll Hindlll Hinfl Hpal Hpall Hphl Hpy188l Hpy188sill Hpy8l Hpy99 HpyAV 19 60 10 13 24 12 45 45 T5 55 23 gl af 5 14 18 126 447 501 718 883 940 976 1420 1821 1904 2043 2667 2955 4343 4823 5459 5661 5678 5757 59 81122 129 346 412 439 447 542 553 634 775 1404 1463 1485 1769 1776 1827 1830 1924 1970 1982 1985 2387 2390 2515 2697 2729 2743 2765 3236 3578 3733 3785 3796 3886 3891 3928 3969 4056 4059 4062 4298 4394 4435 4449 4550 4660 4769 5056 5285 5380 5407 5746 6074 6280 6283 6348 6491 6646 1272 c 1638 c 2204 c 3481 c 4148 4173 4718lc 5361 5648 5829 2695 3254 5920 5577 421 c 436 c 1187 c 1316 1705 c 2387 2387 c 3730 c 3730 4295 c 4322 c 4547 c 5407 3 657 2118 3246 3629 3906 6214 6666 6677 25 38 59 971 2791 2799 3827 6448 3 124 145 423 438 446 657 882 939 1189 1318 1418 1707 1819 2118 2389 2665 2954 3096 3246 3571 3577 3586 3629 3732 3906 4297 4324 4549 4822 5409 5676 5756 6214 6648 6666 6677 2025 4020 5819 76 c 117 322 588 934 992 c 2724 4534 4710 5268 5998 c 6576 c 11 24 37 58 80 318 541 824 826 828 970 1074 1534 1575 1891 1989 2696 2 2
28. 5630 c 5754 c 65 776 c 1174 c 1319 1331 1379 1460 1571 1661 1739 1781 1832 2919 3783 c 4250 6188 c 65 777 c 1175 c 1319 1331 1379 1460 1571 1661 1739 1781 1832 2919 3784 c 4250 6189 c 1369 5262 c 4043 4963 6490 200 334 941 1164 1978 2442 2793 3631 3685 5607 5942 6195 3378 3450 2705 3583 5682 6700 207 706 806 1285 1414 1663 3770 3863 4635 5132 6378 65 724 c 150 c 532 1850 c 3509 c 3773 5760 1981 55974512 763 1689 1929 1978 c 2047 2129 5731 112 c 1848 1866 672 4200 c 5196 6064 6305 c 6603 2946 4128 2632 4617 22 35 845 968 1003 3824 4526 4878 5260 468 c 5734 29 280 424 481 644 940 1243 1273 1413 1576 1600 1655 2013 2334 2381 3241 3342 3414 3537 3572 3581 3630 3987 4256 6528 1056 3855 5504 6555 6482 2985 c 1346 c 1620 1635 1734 2066 2225 2476 c 3168 c 3204 3386 3458 3785 c 4040 c 4122 4186 4256 c 4461 4649 c 4739 5102 c 5347 5542 c 6594 c 126 1839 c 1857 c 3279 c 3581 c 4478 c 4652 5313 5830 c 5996 c 6405 c 34 c 1275 3626 414 c 698 c 2043 3351 3423 4328 c 6384 1373 c 1470 1794 2390 2676 3733 5282 5431 6354 806 1251 1854 2025 3937 4127 4655 5152 5217 6378 18 51 48 226 286 355 455 522 525 889 946 1414 1577 1927 27 27 3053 5558 5805 4549 4762 6210 6489 6655 6673 25 9003 97UM Rev A 2005 Chapter 11 38 Enzyme t of cuts Positions BSmAI BsmBl BsmFI Bsml Bsp1286l Bsp24l BspCNI BspHI BspMI BsrBl BsrDi BsrFl BsrGl Bsrl BssHII BssSl BstAP
29. 72 1984 1987 2589 2392 2517 2699 27 51 2745 2 67 52356 5580 35 55 35 87 3798 5888 3893 3930 3971 4058 4061 4064 4300 4396 4437 4451 4552 4662 4771 5058 5287 5382 5409 5748 6076 6282 6285 6350 6493 6648 806 1458 2456 5381 5401 c 33 54 85 126 409 421 505 593 824 826 1368 1401 1449 1773 1780 1979 2215 2647 2667 2786 2800 2843 3357 3376 3429 3448 3718 3904 4123 4189 4195 4223 4227 4268 4272 4326 4674 5687 6078 6638 6675 298 c 1352 c 1616 1631 1760 1811 2959 4113 4118 4262 c 4516 4457 4544 c 5147 5548 c 97 278 382 391 1748 1896 1934 2637 2641 2677 3231 3999 4077 4158 4167 4245 4622 5121 5157 5174 5432 5478 5496 5837 5942 5954 6032 6040 6051 6126 6704 25 9003 97UM Rev A 2005 Chapter 11 39 Enzyme ofcuts Positions Cjel CJePI Clal Csp6l CVIAI Cvi JI Ddel Dpnl Dral Dralll Drdl Eael Eagl Earl Ecil EcoA II Eco5 MI EcoHl ECoICRI ECONI EcoO109l EcoRV EsaBC3l Fall Fatl Faul 13 13 128 14 31 13 16 e WN D no 22 2 17 173 c 506 1202 c 2089 3292 c 3387 c 3625 4868lc 6027 6091 c 6127 6205 6646 c 173 c 600 c 682 c 1202 c 2009 c 2091 c 2378 2629 c 4868 c 5435 5907 6027 6140 c 2656 4605 810 1029 1222 1675 1955 5281 4129 4642 5518 625 1244 1478 1508 1703 1898 1943 2090 2404 3242 5575 5447 5558 5695 4040 4226 4257 4285 4772 4856 4961 5554 5590 5468 5478 5969 6689 5 124 145 555 548 411 419 425 429 458 446 487 555 595 65 677 698 863 882 908 959 950
30. 83 c 3561 c 3567 c 3591 3597 3604 c 3607 c 3619 c 3739 c 3875 c 4232 c 4425 4774 c 4833 5427 c 5633 c 5780 5861 6261 6511 c 6585 3906 104125292529 2590 27 56 5007 3105 5122 5155 51453156 3679 4668 4849 5221 5586 5625 5860 5913 5927 5932 5984 1245 1275 1425 1602 1731 4261 4543 4582 5029 5388 5547 31 121 128 1984 3306 3930 4698 5164 6105 6350 28 58 84 255 293 420 435 588 630 774 783 869 1556 1596 1409 1453 1462 1969 1978 1995 2675 2705 2737 2 39 2781 2808 2838 3375 3447 3498 3577 3583 3815 3899 3922 4061 4067 4184 4220 4267 4534 4630 5682 6070 6642 6690 2843 4326 22 35 968 3824 13 105 320 350 425 443 1274 1928 2382 2383 2642 3828 3988 4728 4763 5264 5615 6311 1243 3241 3537 4256 2841 4324 197 7 628 1247 1481 1511 1706 1901 1946 2093 2407 3245 3378 3450 3541 5698 4043 4229 4260 4286 4775 4859 4964 5557 9393 5471 5481 5972 6692 23 36 499 705 716 717 790 881 969 1113 1223 1282 1820 2042 2874 2886 2907 3282 3348 3420 3825 3860 4620 4913 5503 5714 5755 5849 6621 6660 25 9003 97UM Rev A 2005 Chapter 11 42 Enzyme t of cuts Positions NII387 7 Notl Nrul Nsil Nspl Pfol Plel Ppil Ppul0l PpuMI Psil Psp03 PspGl Pss Pstl Pvul Pvull RleAl Rsal Rsrl Sacl Sacll Sall SanUl Sap Sau96l Scal ScrF Sell SexAl SfaNI Sfcl Sfil Sfol Siml Smal Smil Spel Sphl SspD5 Ww dq qe Ew ope wo Ee NR Ww Rr OWN n0 01 on B P9 p BG rm WO 32 ef 23 565 1079 2385 2587 eels 3380
31. Amersham e Biosciences Part of GE Healthcare G1S Cell Cycle Phase Marker Assay User Manual 25 90035 97UM Secondary information 25 9003 97 Screening Applications 25 9003 98 Research Applications 25 9003 99 6 month evaluation 25 9004 00 12 month evaluation 25 9004 01 Non profit research Oh G e x i BL 1 1h30m early G 2h30min G Front cover Time lapse images of an asynchronous population of U 2 OS cells exhibiting stable expression of the G1S Cell Cycle Phase Marker G1S CCPM After mitosis and cell division lasting approximately 1 hour there is a phase of 2 5 hours where the sensor exhibits predominantly nuclear distribution Rapid changes in sub cellular distribution associated with this phase are indicative of high Cdk2 cyclin E activity The sensor then demonstrates a phase of progressive export from the nucleus over the following 10 14 hours and a phase of 2 5 hours when it is exclusively cytoplasmic The length of each of these phases correlates with the reported lengths of M G4 S and G phases for rapidly dividing U 2 OS cells Elapsed time and phase are indicated on each image Images were acquired on the IN Cell Analyzer 3000 GE Healthcare 25 9003 97UM RevA 2005 2 Contents 1 Introduction 1 1 The Cell Cycle 1 2 Cell Cycle Phase Markers CCPM 1 2 1 Human helicase B 1 2 2 Ubiquitin C promoter 1 2 3 The G1S CCPM sensor 1 5 Applications in drug discovery 2 Licensing
32. Correlation between sub cellular distribution of G1S CCPM and DNA complement 6 Vector use details 6 1 General guidelines for vector use 6 2 Transfection with pCORON1002 EGFP C1 PSLD 6 2 1 FUGENE 6 Transfection Reagent protocol 6 3 Stable cell line generation with pCORON1002 EGFP C1 PSLD 7 Quality control 7 1 pCORON1002 EGFP C1 PSLD expression vector 7 2 Cell cycle position reporting cell line 8 Troubleshooting guide 8 1 Troubleshooting 25 9003 97UM Rev A 2005 Pagefinder 4 16 17 17 17 18 18 19 19 19 19 19 20 20 21 21 22 25 25 24 24 25 25 el 28 28 29 50 32 32 32 Se 32 53 23 55 54 54 9 References 9 1 References 10 Related products 10 1 Related products 11 Appendix 11 1 Restriction map of PCORON1002 EGFP C1 PSLD 25 9003 97UM Rev A 2005 Pagefinder 55 53 56 56 31 1 Introduction 1 1 The Cell Cycle Eukaryotic cell division Figure 1 1 proceeds through a highly regulated cell cycle comprising the consecutive phases gap1 64 synthesis S gap 2 G5 and mitosis M In a normal cell progress from one phase to the next and within phases is controlled by the activation and deactivation of a series of proteins that constitute the cell cycle machinery Signal transduction pathways couple the detection of DNA damage to the cell cycle machinery at specific checkpoints whilst additional pathways couple the cell cycle
33. I BstBl BstF5l BStKTI BStNI BstUI Bstxl BstY Btgl BtgZl BthCl Btsl Cac8l Cdil Chal 8 10 14 ll 15 60 41 iks 31 468 c 2023 c 2309 c 3675 4763 4805 c 4958 c 5734 4763 4805 c 199 234 c 470 728 1994 c 3324 c 3396 c 3460 c 3975 4507 2451 2544 c 207 706 806 1231 1285 1414 1663 1854 2025 2875 3770 3863 3937 4127 4189 4635 5132 5217 6378 205 1234 4900 5994 c 125 1840 c 1858 c 3280 c 3582 c 4479 c 4651 5312 5831 c 5997 c 6406 c 4855 4960 5968 894 3711 c 4092 4542 227 c 449 2802 4437 4491 c 4958 1970 1979 4057 5566 5740 c 889 1595 2841 4143 4324 5715 1954 156 c 212 527 746 1856 c 3035 3515 c 3768 3969 5155 5325 c 5594 5637 5755 6161 6273 c 6286 c 822 824 4221 1425 c 4416 c 4824 c 5131 6515 55 5 5447 4506 1285 c 1651 c 2217 c 3494 c 4141 4166 4731 c 5354 5641 5822 96 277 581 590 1747 1895 1955 2656 2640 2676 5250 5998 4076 4157 4166 4244 4621 5120 5156 5175 5451 5477 5495 5856 5941 5955 6051 6039 6050 6125 6703 1290 1415 1527 1602 1656 2108 2556 5544 5599 5416 4211 6529 6542 6663 9 531 5310 5653 305 824 026 15 5 1691 2215 2710 2 42 2762 3138 3225 3890 4191 4223 4624 4704 4807 4809 4909 5241 5 54 6064 6645 4545 2741744 3227 3995 4241 4618 5153 5170 5938 5950 6036 6047 6700 29 280 1243 2013 3241 3537 4256 1366 2941 c 4095 c 4276 4439 c 61 83 124 131 348 414 441 449 544 555 636 777 1406 1465 1487 17711778 1829 1832 1926 19
34. M assay when multiplexed with Cell Proliferation Fluorescence Assay Cells were incubated in the presence of Nocodazole for 16 hours prior to BrdU incorporation fixation antibody labelling and imaging on the IN Cell Analyzer 3000 Analysis was performed using the G1S Cell Cycle Trafficking Analysis Module and an EC of 163nM was determined based on the percentage of cells in G phase of the cell cycle Figure 5 17 Nocodazole dose response curve 16 hours for fixed cell G1S CCPM assay multiplexed with Cell Proliferation Fluorescence Assay An EC of 163nM Nocodazole 96 of cells in G was calculated from the dose response curve Mean SD n 8 replicates per dose R 0 84 Jequunu 92 a G2 Cell number 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 Nocodazole M 5 5 5 3 Imaging the G1S CCPM assay multiplexed with Cell Proliferation Fluorescence Assay on the IN Cell Analyzer 1000 Example images demonstrating multiplexing of a fixed cell G1S CCPM assay with the Cell Proliferation Fluorescence Assay imaged on the IN Cell Analyzer 1000 are Figure 5 18 Fixed cell G1S CCPM shown in Figure 5 13 and Figure 5 18 assay green multiplexed with Cell A C Proliferation Fluorescence Assay red imaged on the IN Cell Analyzer 1000 Cells imaged in D PBS A no treatment control cells B cells after 24 hours incubation with 250uM Roscovitine and C cells after 16 hours incubation with 1uM Nocodazole Images shown are
35. SO This can be used as the top concentration if preparing a dose response concentration range 100 x concentrated working solution in DMSO should be diluted 1 in 33 with complete Assay medium to achieve 3 x concentrated solution in 3 DMSO for use in the assay Roscovitine Prepare 25mM Roscovitine 100 x concentration working solution in DMSO by adding 564ul of DMSO to 5mg Roscovitine Vortex mix well to ensure thoroughly dissolved This stock solution can be dispensed into smaller volume aliquots and stored at 15 C to 309C This can be used as the top concentration if preparing a dose response concentration range 100 x concentrated working solution in DMSO should be diluted 1 in 33 with complete Assay medium to achieve 3 x concentrated solution in 3 DMSO for use in the assay e 2xPBS Prepare 2 x concentrated PBS by dissolving 4 tablets of PBS in 400ml of distilled water Mix well to ensure thoroughly dissolved sterile filter before use e Hoechst 33342 Prepare a 100mM stock solution by adding 1 62ml of distilled water to 100mg of Hoechst 33342 Vortex mix well to ensure thoroughly dissolved This stock solution can be dispensed into smaller volume aliquots and stored at 15 C to 30 C Prepare a 10mM working solution by adding 900ul of distilled water to 100ul of 100mM Hoechst 33342 Vortex mix well This working solution can be dispensed into smaller volume aliquots and stored at 15 C to 30 C e Fixing solution Prepare 2 Form
36. aldehyde 2uM Hoechst 33342 in PBS by adding 6ml of 4 Formaldehyde to 6ml of 2 x PBS and 2 4ul of 10mM Hoechst 33342 Mix well pre incubate to 37 C prior to use and use immediately Do not Store 5 1 3 Cell thawing procedure Two cryo vials each containing 1 x 106 cells in 1 ml of cryopreservation medium are included The vials are stored frozen in the vapor phase of liquid nitrogen 1 Remove a cryo vial from storage 2 Thaw the cells by holding the cryo vial in a 37 C water bath for 1 2 minutes Do not thaw the cells by hand at room temperature or for longer than 3 minutes as this decreases viability 3 Working aseptically in a Class II cabinet wipe the cryo vial with 70 v v ethanol and transfer the cells immediately to a T75 flask Add 1ml of Assau medium without Geneticin G418 dropwise to the cells Add a further 18ml of Assay medium Incubate at 37 C 5 CO 95 humidity Once cells are attached 24 hours media can be changed for Growth medium containing Geneticin G418 5 1 4 Cell sub culturing procedure Incubation 5 CO 37 C 95 humidity Split ratio 1 5 to 1 10 twice a week The cells should be subcultured when they reach 70 to 90 confluence All reagents should be warmed to 37 C 1 Aspirate the medium from the cells and discard 2 Wash the cells with 10ml PBS Take care not to damage the cell layer while washing but ensure that the entire cell surface is washed 3 Aspirate the PBS from the
37. arrested at the M phase checkpoint will be removed by fixation and washing steps leaving only G phase arrested cells Apoptotic and necrotic cells may also be removed by wash steps presenting difficulties in the differentiation of cytotoxic and cytostatic effects In addition to the above effects the user should always be aware of the temporal nature of the mechanism detected by the sensor The G1S CCPM indicates the cellular activity of the Cdk2 cyclin E complex and translocates maximally in U 2 OS cells in late G phase prior to S phase entry when this complex is most active Peak activity of the Cdk2 cyclin E complex may vary temporally or be completely absent in certain cell types Drugs such as Mimosine that arrest cells close to the G S boundary or in very early S phase after the peak of Cdk2 cyclin E activity will produce a phenotype in which the sensor has already undergone significant cytoplasmic relocation 5 5 7 Correlation between sub cellular distribution of G1S CCPM and DNA complement Information contained within the nuclear marker channel is often ignored when analysing images from a cell based assay but a considerable amount of information can be made available with minimal effort Hoechst 33342 is a minor groove binder at AT rich regions of DNA and the lack of binding to RNA makes it an extremely effective nuclear marker in cell based assaus If experimental and imaging conditions are controlled to avoid saturation and c
38. cell G1S CCPM assay green multiplexed with Cell Proliferation Fluorescence Assay red imaged on the IN Cell Analyzer 3000 Cells imaged in D PBS A no treatment control cells B cells after 24 hours incubation with 250uM Roscovitine and C cells after 16 hours incubation with 1 uM Nocodazole Images shown are 1 5th of the original image generated by the IN Cell Analyzer 3000 Figure 5 16 shows a Roscovitine dose response curve for the G1S CCPM assay when multiplexed with the Cell Proliferation Fluorescence Assay Cells were incubated in the presence of Roscovitine for 24 hours prior to BrdU incorporation fixation antibody labelling and imaging on the IN Cell Analyzer 3000 Analysis was performed using the G1S Cell Cycle Trafficking Analysis Module and an EC of 34uM was determined based on the percentage of cells in G phase of the cell cycle Figure 5 16 Roscovitine dose response curve 24 hours for a fixed cell G1S CCPM assay multiplexed with the Cell Proliferation Fluorescence Assay An EC of 34uM Roscovitine of cells in G was calculated from the dose response curve Mean SD n 8 replicates per dose R2 0 81 t Jequinu 29 96 G1 Cell number 70 65 60 55 50 45 40 35 30 Roscovitine M 5 5 5 2 G1S CCPM fixed cell assay multiplexed with Cell Proliferation Fluorescence Assay to produce Nocodazole dose response Figure 5 17 shows a Nocodazole dose response curve for the G1S CCP
39. ces 25 9001 89 e Hoechst 33342 trihydrochloride trinydrate Fluoropure grade Molecular Probes H 21492 e 4 w v Formaldehyde ACCUSTAIN Formalin solution 10 neutral buffered Sigma HT50 1 2 e Phosphate Buffered Saline Tablets Sigma P 4417 e Water Analar BDH Prod 102923C e 96 well Packard Viewplates 6005182 e Standard tissue culture plastic ware including tissue culture treated flasks T flasks and cryo vials 5 1 2 Reagent preparation NOTE the following reagents are required but not supplied e Growth medium McCOYS 5A medium modified supplemented with 10 v v FBS 1 v v Penicillin Streptomucin 1 v v L Glutamine and 1 v v Geneticin working concentration 500ug ml 1 e Assay medium McCOYS 5A medium modified supplemented with 10 v v FBS 1 v v Penicillin Streptomucin and 1 v v L Glutamine e Cryopreservation medium McCOYS 5A medium modified supplemented with 1096 v v FBS 196 v v Penicillin Streptomucin 196 v v L Glutamine and 1096 v v DMSO e Nocodazole Prepare a 1mg ml stock solution equivalent to 3 3mM in DMSO by adding 10ml of DMSO to 10mg of Nocodazole Vortex mix well to ensure thoroughly dissolved This stock solution can be dispensed into smaller volume 25 9003 97UM Rev A 2005 Chapter 5 14 aliquots and stored at 15 C to 30 C Prepare 100UM Nocodazole 100 x concentration working solution in DMSO by adding 30ul of the 3 3mM stock solution to 970ul of DM
40. e Images acquired on IN Cell Analyzer 3000 oO CO CO N gt O Co NO IA O l l l l l Nuc Cyt ratio green 0 2 0 0 Time hours Figure 1 4 Graph of sub cellular distribution nuclear cytoplasmic ratio in green channel of G1S CCPM sensor through one complete cell cycle time lapse images taken at 30 minute intervals over 25 hours on the IN Cell Analyzer 3000 In a single cell the sub cellular distribution of the sensor exhibits 4 distinct phases that correlate with reported lengths for M G4 S and G phases in U 2 OS cells 25 9003 97UM Rev A 2005 Chapter 1 8 1 3 Applications in drug discovery Accurate determination of the position of a particular cell within the cell cycle is essential in assessing the effects of environmental factors upon a cell and the CCPMs can be used in e drug screening to uncover and characterise novel anti cancer drugs that arrest cell proliferation e toxicology to establish whether a lead compound has adverse effects upon the rate or control of the cell cycle e a complex assay to determine the effect of cell cycle position on a separate process such as gene expression measured by a reporter gene assay or a signal transduction measured by a protein translocation event Kinetic visualisation of the CCPMs can provide continuous real time live cell images allowing the progress of individual cells through the cell cycle t
41. e Assay Cells were incubated in the presence of Nocodazole for 16 hours prior to BrdU incorporation fixation antibody labelling and imaging on the IN Cell Analyzer 1000 The images obtained from the IN Cell Analyzer 1000 were converted using the IN Cell Image Converter 123 and analysis was performed using the G1S Cell Cycle Trafficking Analysis Module Based on the percentage of cells in G phase of the cell cycle an EC59 of 155nM was determined this value compares directly with that obtained on the IN Cell Analyzer 3000 see section 5 5 5 2 Figure 5 20 Nocodazole dose response curve 16 hours for the G1S CCPM assay multiplexed with Cell Fluorescence Proliferation fixed cell assay imaged on the IN Cell Analyzer 1000 converted and analysed using the G1S Cell Cycle Trafficking Analysis Module An EC of 155nM d ss Nocodazole 96 of cells in G was Cell number calculated from the dose response 90 85 80 75 70 65 460 55 curve Mean SD n 8 replicates per dose R2 0 74 Jequunu 923 Nocodazole M 5 5 6 Characterisation of G1S CCPM with inhibitors of the cell cycle The phase specific sub cellular localisation of the G1S CCPM sensor has been characterised further using chemical and siRNA based arrest Figure 5 21 Agents known to effect arrest in G4 phase Olomoucine Roscovitine serum starvation and double Thymidine block and siRNAs against minichromosome maintenance proteins MCM cyclin E retinol binding pro
42. ells 2 Add 50ul of 3 DMSO control solution to appropriate negative DMSO control wells 3 Add 50ul of appropriate 3 x concentrated Roscovitine solution in 3 DMSO see section 5 1 2 to appropriate wells 4 Return plate to incubator and incubate at 37 C 5 CO 95 humidity for 24 hours 5 2 2 2 G2M cell cycle synchronization using Nocodazole 1 Add 50ul of Assay medium only to appropriate negative control wells 2 Add 50ul of 3 DMSO control solution to appropriate negative DMSO control wells 3 Add 50ul of appropriate 3 x concentrated Nocodazole solution in 3 DMSO see section 5 1 2 to appropriate wells 4 Return plate to incubator and incubate at 37 C 5 CO 95 humidity for 16 hours Day 4 Ensure all solutions are pre warmed to 37 C prior to use unless otherwise stated Incorporation and detection of BrdU using the Cell Proliferation Fluorescence Assay can be a useful indicator of DNA replication and S phase cells In addition BrdU can be used to verify cell cycle arrest indicated using the G1S CCPM The following protocol describes the option to pulse label G1S CCPM U 2 OS cells with BrdU reagent for 1 hour prior to washing and fixation If BrdU labelling is required proceed with steps 1 9 below If no BrdU labelling is required proceed through steps 3 8 below prior to imaging according to section 55 1 Add 50yl of Assay medium only to control wells controls in the absence of BrdU provide an indication
43. ertain assumptions 25 9003 97UM RevA 2005 Chapter 5 30 Figure 5 21 Effect of phase specific chemical and siRNA induced cell cycle arrest on U 2 OS cells exhibiting stable expression of the G1S CCPM Cells were untreated treated with drugs for 24 hours or transfected with siRNAs Dharmacon SIARRAYT at 25nM in Lipofectamine 2000 Invitrogen for 4 hours followed by a media change and further incubation for 20 hours post transfection Cells in top figures also indicate BrdU incorporation red Fixed cells were imaged on the IN Cell Analyzer 1000 GE Healthcare and where appropriate blocks were confirmed using propidium iodide staining and flow cytometry are made regarding the relationship between fluorescent emission and binding one can relate the integrated fluorescent intensity of the nucleus with DNA complement The nuclear mask can also provide data regarding cell number nuclear area nuclear shape and nuclear fragmentation which can be useful indicators of toxicity apoptosis and aberrant mitosis or cell division Please refer to the IN Cell Analyzer 1000 and IN Cell Analyzer 3000 Analysis Modules or contact your local GE Healthcare representative for further information The correlation between G1S CCPM phenotype and DNA complement provides further evidence that the cytoplasmic relocation of the sensor is cell cycle related and occurs prior to DNA replication Figure 5 22 Dual analysis of DNA complement and the pheno
44. eservation of samples for re interrogation optically clearer images and more sensitive assays since fluorescent background due to media components is reduced interrogation of alternative markers via immuno labelling measurement of cellular DNA complement since the integrated Hoechst or propidium iodide intensity total nuclear intensity is proportional to DNA complement assuming that the system is not saturated However fixation has a number of disadvantages that can be addressed with a live cell end point imaging format Washing and fixation steps can result in a loss of poorly adhered cells including mitotic apoptotic and dead cells These and other fixation artifacts can affect assay results and cause mis interpretation of drug effects The discrimination of live mitotic cells from cells undergoing rounding due to necrosis is relatively simple in live cell format by addition of propidium iodide 10uM and Hoechst 2uM immediately prior to imaging In addition live cell imaging even in the presence of a DNA binding nuclear marker permits short term temporal analysis but such markers affect cell cycle dynamics over longer exposure times True kinetic imaging can be used to obtain a large amount of temporal information and produce time lapse movies over long time periods 48 hours However automatic analysis modules are not currently available for cells that do not contain a nuclear marker In addition photobleaching and phototoxicity can resu
45. ge gt 20 to determine homogeneity and consistency of expression and biological response 5 Secondary cloning if required repeat 1 4 on primary clone 7 Quality control 7 1 pCORON1002 EGFP C1 PSLD expression vector The pCORON1002 EGFP C1 PSLD vector is supplied in TE buffer 10mM Tris 1mM EDTA pH 8 0 at 250ug ml The vector has the characteristics outlined in Table 7 1 and Table 7 2 Property Value Limits Measurement method Concentration 250ug ml UV Absorbance 9 260nm in water Purity Minimal Aseo Asgo Between UV Vis contamination X ratio 1 8 2 2 Absorbance of the DNA 260nm construct by and 280nm RNA or protein Expected The restriction Agarose gel restriction digests should electrophoresis pattern give fragments of the sizes Shown in Table re Table 7 1 Quality control information for the pCORON1002 EGFP C1 PSLD expression vector Restriction enzyme of cuts Expected size of fragments bp Nhel 1 6 04 Hindlll 2 24531 4273 Ncol 4 296 19 1998 3691 Bglll 1 6 04 Psil 3 564 1643 4497 Table 7 2 Expected restriction pattern for the pCORON1002 EGFP C1 PSLD expression vector 7 2 Cell cycle position reporting cell line The cell cycle position reporting cell line is supplied at a concentration of 1 x 106 cells per ml in fetal calf serum containing 1096 v v DMSO Assays performed in the development of the G1S CCPM assay have been carried out in a final concentration of 196 DMSO Users
46. i Maelll Mbol Mboll Mfel Mlyl Mmel MnlI Nael Nar Neil Ncol NgoMIV Nhel Nlalll NlalV 14 16 Zi 46 23 51 Ze 54 22 11 10 46 18 27 50 2034 2053 2690 2972 3669 4204 4320 4428 4757 4792 5360 5875 6188 6658 64 723 845 1309 1522 1693 2835 2945 2988 3000 3940 4127 4878 5198 5571 5987 804 872 1390 1451 1775 2449 2517 2544 5569 3378 3441 3450 3565 3641 3716 3875 4050 4064 4633 4771 5130 5374 5462 5655 5745 6080 6376 29 59 85 234 294 421 436 589 631 775 784 870 1337 1397 1410 1454 1463 1970 1979 1996 2676 2706 2738 2740 2782 2809 2839 3376 3448 3499 3578 3584 3816 3900 3923 4062 4068 4185 4221 4268 4535 4631 5683 6071 6643 6691 21 54 967 3823 1225 3284 23 36 57 969 2789 2797 3825 6446 193 391 733 841 846 1427 1916 2075 2102 2500 2756 2768 3944 4250 4751 5139 5327 5480 5538 5869 6152 6268 6331 93 274 378 387 1744 1892 1930 2633 2637 2673 3227 3995 4073 4154 4163 4241 4618 5117 5153 5170 5428 5474 5492 5833 5938 5950 6028 6036 6047 6122 6700 588 1028 c 1487 c 1532 c 1535 c 1730 2030 2113 c 2161 c 2287 c 2671 2807 c 3647 c 4185 4395 4475 c 5018 5127 5205 5960 6031 c 6183 c 999 133 c 190 c 573 3003 3010 c 4489 c 5810 6312 c 297 1 c 6295 c 6479 c 14 35 52 55 231 c 264 c 581 c 593 c 647 c 840 97 7 c 982 c 986 993 c 1253 c 1334 c 1340 c 1434 1571 c 1583 c 1634 c 1754 c 2002 2031 c 2108 c 2128 c 2129 2575 c 2615 2655 c 2919 3259 c 3267 32
47. ion 9 References 9 1 References 1 Humphrey T Brook G 2005 Methods in Molecular Biology Cell Cycle Control mechanisms and protocols Humana Press Totowa NJ USA And chapter and citations therein 2 Taneja P et al 2002 J Biol Chem 277143 40853 61 3 Gu J et al 2004 Mol Biol Cell 15 7 3320 32 4 Schorpp M et al 1996 Nucleic Acids Res 24 9 1787 8 5 Lois C et al 2002 Science 295 5556 868 72 6 Weis K 2003 Cell 112 441 451 7 Ponten J et al 1967 Int J Cancer 2 434 447 8 Heldin CH et al 1986 Nature 319 511 514 9 Raile K et al 1994 J Cell Physiol 159 531 541 10 Diller et al 1990 Mol Cell Biol 10 5772 5781 11 Stott FJ et al 1998 EMBO 17 5001 5014 12 Bunz F et al 1998 Science 282 1497 1501 13 Flatt PM et al 2000 Mol Cell Biol 20 4210 4223 25 9003 97UM Rev A 2005 Chapter 9 35 10 Related products 10 1 Related products Please consult your local GE Healthcare representative Product Name G2M Cell Cycle Phase Marker Cell Proliferation Fluorescence Assay GFP assays GFP PLCS PH domain Assay GFP Rac1 Assay GFP MAPKAP k2 Assay AKT1 EGFP Assay EGFP 2xFYVE Assay EGFP STAT3 Assay EGFP NFATc1 Assay EGFP SMAD2 Assay IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module G2M Cell Cycle Trafficking Analysis Module IN Cell Image Converter 123 IN Cell Analyzer 1000 IN Cell Analyzer 1000 Morphology Analysis Module 25
48. lt from repeated exposure of the sensor and cells to high energy light sources and therefore should be avoided 5 2 2 G1S CCPM fixed cell assay procedure The following sections describe a fixed cell assay procedure and the use of the G1S CCPM assay to assess cell cycle synchronization arrest with Roscovitine and Nocodazole Fixed cell assays permit detection of additional markers and as an example a method for the co detection of DNA replication using the Cell Proliferation Fluorescence Assay has been provided please refer to Cell Proliferation Fluorescence Assay manual Product code 25 9001 89 for further details 25 9003 97UM Rev A 2005 Chapter 5 17 Day 1 To ensure that the population is in logarithmic growth phase cells should be sub cultured prior to use e g ratio of 1 2 in two T 175 flasks Actively growing cells will be ready for seeding into microwell plates the following dau Day 2 Seed 5000 cells per well in Assay medium 100ul per well into 96 well microplates as described in section 5 1 5 Incubate the cells for 24 hours at 37 C 5 CO 95 humidity before starting the assay It is essential that the number of cells per well in the assay plates is consistent in order to minimise assay variability Day 3 Ensure assay media control and compound solutions are pre warmed to 37 C prior to use 5 2 2 1 G1S cell cycle synchronization using Roscovitine 1 Add 50ul of Assay medium only to appropriate negative control w
49. lthcare 25 9001 89 Section 5 5 5 allows for precise resolution of cells in S phase and can be used to determine effects of agents upon DNA replication Multiparametric analysis of multicolour images can be employed to correlate results of the CCPM assay DNA complement cell number replicative activity and indicators of nuclear and cellular morphology The current G1S CCPM can be used in a cell based assay to screen for cell permeable inhibitors of the Cdk2 cuclin E complex see Chapter 5 25 9003 97UM Rev A 2005 Chapter 1 2 Licensing considerations 2 1 Licensing considerations Use of this product is limited in accordance with the terms and conditions of sale of the product code purchased 25 9003 97 for Screening Applications 25 9003 98 for Research Applications 25 9003 99 for 6 month evaluation 25 9004 00 for 12 month evaluation and 25 9004 01 for Non profit research The G1S Cell Cycle Phase Marker Assay is the subject of international patent application numbers PCT GB2005 002876 PCT GB2005 002884 and PCT GB2005 002890 in the name of Amersham Biosciences and Vanderbilt University The G2M Cell Cycle Phase Marker Assay is the subject of patent applications AU 2002326036 CA 2461133 EP 02760417 2 IL 160908 JP 2003 534582 and US 10 491762 in the name of Amersham Biosciences and Cancer Research Technology The IN Cell 1000 is the subject of US patents 6 563 653 amp 6345115 and US patent application number 10 514925 t
50. machinery to the detection of external factors This elaborate and ordered control of the cell cycle and its checkpoint mechanisms ensures tight regulation in response to cellular and environmental factors and maintains genomic integrity by arresting progress or inducing destruction of aberrant cells Figure 1 1 The distinct phases of the cell cycle are controlled through the interaction of cyclins cyclin dependent kinases and their inhibitors Cells that are quiescent or resting are in Gj phase A G phase cell can be stimulated to re enter the cell cycle through exposure to a range of external factors such as mitogens via the mitogen activated protein kinase cascade During G phase cells prepare for DNA replication and pass through a checkpoint for DNA integrity prior to S phase In S phase the genome is replicated and centrosomes are duplicated Once DNA replication has been completed the cell enters G phase where proteins required for mitosis are synthesised and final checks on the integrity of the DNA are made Mitosis involves chromosome condensation alignment and sister chromatid migration which occur prior to the physical division of the cell into two daughters a process known as cytokinesis 25 9003 97UM RevA 2005 Chapter 1 1 2 Cell Cycle Phase Markers CCPM The Cell Cycle Phase Markers CCPM are non perturbing fluorescent protein based sensors that provide an indication of the cell cycle status of individual cells i
51. mperature 5 Add transfection mixture directly to the cells dropwise without changing the medium and mix by swirling gently Day 3 Change media to a complete media without washing the cells Day 3 4 Cells are ready for use Stable cell lines may be obtained by sub culturing 1 10 and selecting for resistant cells using Geneticin G418 antibiotic 25 9003 97UM RevA 2005 Chapter 6 6 3 Stable cell line generation with pCORON1002 EGFP C1 PSLD The process of establishing stable cell lines involves a large number of variables many of which are cell line dependent Standard processes see below and guidelines for the generation of stable cell lines are widely available specific methods are beyond the scope of this manual 1 Isolate 10 60 primary clones using FACS or conventional cloning ring methods 2 Characterise clones at early passage using flow cytometry to determine expression levels and fluorescence microscopy to determine morphology and distribution of Sensor 3 For clones n 5 10 that are deemed suitable characterise physiological relevance of biological response determine temporal distribution of sensor with cell cycle correlate N C ratio with DNA concentration correlate distribution with other markers of cell cycle e g BrdU analyse growth rate characterise sensor distribution after treatment with agents that block cell cycle 4 For clones that are deemed suitable from step 3 characterise at late passa
52. n an asynchronous population They can be used in live cells with kinetic imaging systems to provide a dynamic non invasive method of monitoring the cell cycle or in end point format with other probes to highlight complex cell cycle related events GE Healthcare has previously demonstrated the use of functional elements from the cyclin B1 promoter and gene in the development of a live cell non perturbing sensor of G and M phases of the cell cycle termed the G2M Cell Cycle Phase Marker G2M CCPM Product code 25 8010 50 The G2M CCPM sensor is switched on in late S phase switched off at the end of mitosis and in the intervening period during prophase translocates from the cytoplasm to the nucleus The current G1S Cell Cycle Phase Marker G1S CCPM has been developed using functional elements from the human helicase B gene and human ubiquitin C promoter Figure 1 2 The G1S CCPM provides a live cell non perturbing sensor of Cdk2 cuclin E activity and is an indicator of G4 and S phases of the cell cycle In conjunction with fluorescence microscopy the G1S CCPM can indicate the phase specific cell cycle position of individual cells within an asynchronous population 1 2 1 Human helicase B The human homolog of helicase B was first reported in 2002 2 The 1087 amino acid protein comprises three regions an uncharacterised N terminal region a central helicase domain containing seven conserved motifs of the helicase superfamily
53. n vapour 4 3 Handling Upon receipt the vector should be removed from the cryo porter and stored at 15 C to 30 C until used The cells should be removed from the cryo porter and transferred to a gaseous phase liquid nitrogen storage unit Care should be taken to ensure that the cells are not warmed if they are not being used immediately 4 3 1 Vector After thawing the DNA sample centrifuge briefly to facilitate full recovery of the contents 4 3 2 Cells Do not centrifuge the cell samples upon thawing 5 Cell assay design 5 1 Culture and maintenance of U 2 OS cell line exhibiting stable expression of the G1S CCPM 5 1 1 Tissue culture media and reagents required The following media and buffers are required to culture maintain and prepare the cells for the assay e McCOYS 5A medium modified Sigma M8403 e Foetal Bovine Serum FBS Australian Origin Sigma F9423 e L Glutamine 200mM Sigma 67513 e Penicillin Streptomycin solution stabilized 10 000 units ml and 10mg ml 1 respectively Sigma P4333 e Trypsin EDTA 1x porcine trypsin 0 59 l 1 and EDTA 4Na 0 29 1 1 HBSS Sigma 13924 e D PBS CaCl MgCl Invitrogen life technologies 14190 094 e G418 disulphate salt solution Antibiotic G418 Sigma G 8168 e Dimethylsulfoxide DMSO HYBRI MAX Sigma D 2650 e Roscovitine Sigma R 7772 e Nocodazole Sigma M 1404 e Cell Proliferation Fluorescence Assay CPFA Amersham Bioscien
54. ng rate of 1 C per min e g Nalgene Mr Frosty Sigma C 1562 e Standard tissue culture reagents and facilities see also section 5 1 1 3 5 Software requirements 3 5 1 IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module Cell cycle status can be determined by measurement of G1S CCPM fluorescence intensity sub cellular distribution and other cellular characteristics from 2 or 5 colour images see section 5 3 1 4 Image analysis outputs for individual cells and the total population include cell number phase classification cell rounding nuclear area nuclear cytoplasmic and cellular intensity values for the blue green and red channel Analyzed data are exported in the form of numerical files in ASCII format These data can be utilized by Microsoft Excel Microsoft Access or any similar packages 3 5 2 IN Cell Analyzer 1000 G1S Cell Cycle Trafficking Analysis Module An analysis module for images acquired on the IN Cell Analyzer 1000 is under development The IN Cell Image Converter 123 see section 3 5 4 is available to facilitate use of the IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module with files produced on the IN Cell Analyzer 1000 see Section 5 5 5 5 for examples of this analysis method Please contact your local GE Healthcare representative for availability 3 5 3 Confocal or epifluorescence microscopes Suitable sub cellular analysis software will be required for analysi
55. ntained throughout S G and M phases whilst mutation of S967 showed this residue to be crucial to the cell cycle phase specific subcellular localisation of the protein Both Cdk2 cyclin A and Cdk2 cuclin E complexes can phosphorylate S967 of helicase B in vitro but Cdk2 cyclin E has been shown to be the predominant complex with ectopically expressed helicase B in asynchronous cell extracts Gu et al 3 concluded that in G4 phase cells S967 of human helicase B is dephoshorulated the NLS is exposed the NES is masked and therefore the protein is predominantly distributed in the nucleus However late in G phase S967 becomes phosphorylated by increasing levels of active Cdk2 cyclin E complex resulting in the unmasking of an NES and nuclear to cytoplasmic translocation of the protein 5967 Walker sites 1 HOHB 027 PSLD Sna PSLD Figure 1 2 Schematic of human helicase B and development of the G1S CCPM sensor containing the phosphorylation dependent subcellular localisation control domain PSLD In late G phase S967 of human helicase B is phosphorylated by the Cdk2 cyclin E complex putative CDK phosphorylation sites are shown in yellow This event is thought to unmask a rev type nuclear export sequence NES resulting in translocation of the endogenous protein from the nucleus to the cytoplasm around the G S boundary The G1S CCPM sensor is a fusion of the helicase B PSLD region to the C terminus
56. o be determined temporallu obviating the need for synchronisation physical intervention or fixation In addition the CCPMs can be run in fixed or live cell end point formats in the presence of nuclear markers and other sensors The images generated with end point CCPM assays provide the opportunity to analyse population and individual cell data regarding the intensity and subcellular localisation of the sensor and other indicators of cellular physiology For example in fixed cell assays the integrated nuclear marker intensity can be used to indicate DNA complement 2n 4n or 8n whilst co analysis and determination of the cell cycle phase using CCPMs can be used to correlate these two parameters Data regarding cell number and nuclear and cellular morphology can also be acquired and correlated with CCPMs to provide an indication of toxicity apoptosis endoreduplication and aberrant mitosis Multiplexing a CCPM with another distinct output provides an opportunity to correlate virtually any cellular event or process for which a probe is available with cell cycle position For example addition of propidium iodide to a live cell end point CCPM assay can be used to stain dead cells since propidium iodide is not live cell permeable Multiplexing a CCPM with a second marker of the cell cycle will permit increased resolution of the progress of individual cells through the cell cycle For example the Cell Proliferation Fluorescence Assay GE Hea
57. o local law which MUST be followed In the case of the UK this is the GMO Contained Use Regulations 2000 Information to assist users in producing their own risk assessments is provided in the Regulations in particular sections 3 3 1 and 3 3 2 Risk assessments made under the GMO Contained Use Regulations 2000 for our preparation and transport of these cells indicate that containment 1 is necessary to control risk This risk is classified as GM Class 1 lowest category in the United Kingdom For handling precautions within the United States consult the National Institute of Health s Guidelines for Research Involving Recombinant DNA Molecules Instructions relating to the handling use storage and disposal of genetically modified materials 1 These components are shipped in liquid nitrogen vapour To avoid the risk of burns extreme care should be taken when removing the samples from the vapour and transferring to a liquid nitrogen storage unit When removing the cells from liquid nitrogen storage and thawing there is the possibility of an increase in pressure within the vial due to residual liquid nitrogen being present Appropriate care should be taken when opening the vial 2 Genetically modified cells supplied in this package are for use in suitably equipped laboratory environment and should only be used by responsible persons in authorised areas Care should be taken to prevent ingestion or contact with skin or clothing
58. of background levels and should be carried out 2 Add 50ul of 1 in 125 diluted Labelling Reagent to wells that are to be labelled with BrdU 1 in 500 final dilution of BrdU labelling reagent and incubate at 37 C 5 CO 95 humidity for 1 hour 3 Gently remove media from all wells harsh washing and agitation during steps 4 11 will detach poorly adhered cells including apoptotic and mitotic cells and can cause mis interpretation of results 4 Wash the cells with gentle addition and removal of 200ul of D PBS 5 Add 100ul of Fixing solution containing nuclear dye in PBS see section 5 1 2 to all wells and incubate in dark at room temperature for 20 minutes 6 Remove Fixing solution from all wells and discard as toxic waste 7 Wash the cells twice with gentle addition and removal of 200ul of D PBS 25 9003 97UM RevA 2005 Chapter 5 18 8 Add 200ul of D PBS to all wells prior to storage 4 C in dark imaging or detection of BrdU 9 To detect incorporated BrdU please refer to Cell Proliferation Fluorescence Assay manual Product code 25 9001 89 for procedure and further details 5 2 3 G1S CCPM live cell end point assay procedure Proceed as Days 1 2 and 3 for the fixed cell assay procedure as described in section 5 2 2 Live cell endpoint and true kinetic imaging on non confocal instruments will require imaging and assay procedures to be optimised for particular imaging systems Background should be reduced to a minimum
59. ogether with other granted and pending family members in the name of Amersham Biosciences Niagara Inc and The IN Cell Analyzer 3000 is the subject of US patents 6 400 487 and 6 388 788 and US patent application number 10 227552 together with other granted and pending family members in the name of Amersham Biosciences Corporation The cell cycle phase marker products are sold under license from Invitrogen IP Holdings Inc formerly Aurora Biosciences Corporation under patents US 5 625 048 US 5 777 079 US 5 804 387 US 5 968 738 US 5 994 077 US 6 054 321 US 6 066 476 US 6 077 707 US 6 090 919 US 6 124 128 US 6 319 669 US 6 403 374 EP 0804457 EP 1104769 JP 3283523 and other pending and foreign patent applications Biolmage A S under patents US 6172188 US 5958713 EP 851874 EP 0815257 and JP3535177 Columbia University under US patent Nos 5 491 084 and 6 146 826 University of Florida under US patents 5 968 750 5 874 304 5 795 757 6 020 192 and other pending and foreign patent applications The IN Cell Analyzer 1000 IN Cell Analyzer 3000 and their associated analysis modules are sold under license from Cellomics Inc under US patent Nos 6573039 5989835 6671624 6416959 6727071 6716588 6620591 6759206 Canadian patent No 2328194 2562117 2 282 658 Australian patent No 730100 European patent 1155304 and other pending and foreign patent applications Rights to use this product as configured are limited to internal use fo
60. ours prior to fixation and imaging on the IN Cell Analyzer 3000 Analysis was performed using the G1S Cell Cycle Trafficking Analysis Module and an EC of 187nM was determined based on the percentage of cells with a G phenotupe m Figure 5 10 Fixed cell assay Nocodazole dose response curve 16 hours for U 2 OS cells exhibiting stable expression of G1S CCPM An EC of 187nM Nocodazole of cells in G J Mean SD n 8 replicates per dose R 0 87 60 50 40 30 Jequunu 92 20 G2 Cell number 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 Nocodazole M 5 5 3 G1S CCPM live cell end point assay Roscovitine dose response Figure 5 11 shows a Roscovitine dose response curve for a live cell end point G1S CCPM assay Cells were incubated in the presence of Roscovitine for 24 hours prior to addition of Hoechst nuclear marker and live cell imaging on the IN Cell Analyzer 3000 Analysis was performed using the G1S Cell Cycle Trafficking Analysis Module and an ECs of 31uM was determined based on the percentage of cells with a G phenotype m Figure 5 11 Live cell assay Roscovitine dose response curve 24 hours for U 2 OS cells exhibiting stable expression of the G1S CCPM An EC of 31uM Roscovitine 96 of cells in G4 was calculated from the dose response curve Mean SD n 8 replicates per dose R 0 90 60 50 40 30 Jequunu 92 20 95 G1 Cell number 7 0 6 5 6 0
61. r screening development and discovery of therapeutic products NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rights are conveyed 25 9003 97UM Rev A 2005 Chapter 2 10 2 2 Legal Amersham and Amersham Biosciences are trademarks of GE Healthcare Limited GE and GE monogram are trademarks of General Electric Company FUGENE is a trademark of Fugent LLC Microsoft is a trademark of Microsoft Corporation FACS is a trademark of Becton Dickinson and Co Hoechst is a trademark of Aventis Geneticin is a registered trademark of Life Technologies Inc ACCUSTAIN and HYBRI MAX are trademarks of Sigma Aldrich SIARRAY is a trademark of Dharmacon INC Lipofectamine is a trademark of Invitrogen Corp 2005 General Electric Company All rights reserved General Electric Company reserves the right subject to any regulatory approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your GE Representative for the most current information and a copy of the terms and conditions http www amershambiosciences com Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 3 Product contents 3 1 Components summary e pPCORON1002 EGFP C1 PSLD expression vector encoding G1S CCPM fusion protein 1 vial containing 10ug DNA Supplied in TE buffer
62. rder to minimize perturbation of host cell systems and produce an assay that is stable and robust Characterisation of a clonal U 2 OS cell line exhibiting stable expression of the G1S CCPM on the IN Cell Analyzer 3000 has demonstrated that the subcellular distribution of the sensor and other cellular characteristics can be used to indicate up to 4 separate phases of the cell cycle see front cover Figure 1 3 Figure 1 4 and Chapter 5 After mitosis and cell division lasting 1 to 1 5 hours there is a phase of 2 5 hours where the sensor exhibits predominantly nuclear distribution Rapid changes in sub cellular distribution associated with this phase are indicative of increasing Cdk2 cuclin E activity The sensor demonstrates a slower progressive export from the nucleus over the following 10 14 hours and a period of 2 5 hours when it is exclusively cytoplasmic The length of each of these phases correlates with the reported lengths of G4 S G gt and M phase for rapidly dividing U 2 OS cells cycle progression phase Early G 1h30m 2n30min 4h 7h30m Figure 1 3 Schematic and time lapse images of G1S CCPM The upper panel shows a schematic of the relative brightness and size of cells and sub cellular compartments for each phase of the cell cycle The lower panel contains live time lapse images times shown on each image of U 2 OS cells exhibiting stable expression of the G1S CCPM passing through one complete cell cycl
63. resentative images of asynchronous U 2 OS cells exhibiting stable expression of the G1S CCPM followed over 24 hours on an Axiovert 100 25 9003 97UM RevA 2005 Chapter 5 22 microscope imaging at 20 minute intervals Elapsed time and phase for a single cell are indicated on each image 300 division o I nuc 250 N C ratio 200 late S 3 late G2 150 GFP fluorescence 4 0 5 a gt a gt 3 6 9 12 15 18 21 Time hr Figure 5 8 Analysis of a single U 2 OS cell and one daughter after division exhibiting stable expression of the G1S CCPM followed over 24 hours on an Axiovert 100 microscope imaging at 20 minute intervals The sensor exhibits progressive clearing from the cytoplasm during S phase and G phase Mitosis and cell division last approximately 1 hour and are followed by a G phase when the sensor is predominantly nuclear for approximately 4 5 hours between the 14 and 19 hour period 5 5 G1S CCPM sensor and assay characterization The publication of Gu et al 3 describes the characterisation of helicase B and motifs within the PSLD Data presented elsewhere in this manual describe the characterisation of the sub cellular distribution of the G1S CCPM sensor during the cell cycle and growth characteristics of the cell line A number of methods have been used to further characterise the G1S CCPM sensor and are described below 5 5 1 G1S CCPM fixed cell assay Roscovitine dose re
64. rogen storage device 5 1 7 Growth characteristics Under standard growth conditions the cells should maintain an average size of 18 20um as measured using a CASY1 Cell Counter and Analyzer System Model TT The doubling time for the U 2 OS cell line exhibiting stable expression of the G1S CCPM has been determined to be 25 5 hours 9596 confidence range 21 33 hours using the CellTiter 968 AQ eous One Solution Cell Proliferation Assay Promega G3582 An equivalent doubling time value of 28 3 hours 95 confidence range 22 37 hours was obtained for the parental U 2 OS cell line 25 9003 97UM RevA 2005 Chapter 5 16 indicating that expression of the G1S CCPM reporter molecule has minimal effects on the length of the cell cycle Figure 5 1 0 5 Figure 5 1 Growth curve of untransfected U 2 OS cells and cells p expressing the G1S CCPM mean and T SD n 6 R2 0 88 for G1S CCPM and S 0 92 for U 2 OS tO Q 02 0 1 m U 20S G1S CCPM 0 0 0 10 20 30 40 50 60 Hours 5 2 Assay set up 5 2 1 General Assay Set up The G1S CCPM assay can be performed in fixed or live cell format as an end point procedure in the presence of a nuclear marker or as a true kinetic procedure It is important to consider the best assay format to address the aims of an experiment The fixed cell format has a number of advantages when compared to live cell end point or kinetic imaging including convenience with respect to assay scheduling and pr
65. ronous populations of U 2 OS cells exhibiting stable expression of the G1S CCPM are presented in Figure 5 4 Cells were seeded onto a Packard Viewplate and imaged every 30 minutes for 48 hours on the IN Cell Analyzer 3000 see section 5 2 4 Figure 5 2 Fixed U 2 OS cells exhibiting stable expression of the G1S CCPM imaged on the IN Cell Analyzer 3000 section 5 2 2 A control cells B 24 hours incubation with Roscovitine 250uM and C 16 hours incubation with Nocodazole 10M Images shown are a 1 5th section of the original image Figure 5 3 Live U 2 OS cells exhibiting stable expression of the G1S CCPM imaged on the IN Cell Analyzer 3000 section 5 2 3 A control cells B 24 hours incubation with Roscovitine 250uM and C 16 hours incubation with Nocodazole 10M Images shown are a 1 5th section of the original image Figure 5 4 Typical kinetic images of asynchronous U 2 OS cells exhibiting stable expression of the G1S CCPM imaged on the IN Cell Analyzer 3000 25 9003 97UM Rev A 2005 Chapter 5 20 Elapsed time is indicated on each image and an individual cell has been marked arrow for tracking purposes In control cells bottom two panels after cell division there is a G phase of 5 hours where the sensor exhibits predominantly nuclear distribution The sensor then demonstrates progressive export from the nucleus indicative of an S phase lasting 14 hours and a phase of 5 hours when it is exclusivel
66. s of images acquired on these microscopes The IN Cell Image Converter 123 is available to facilitate analysis of images generated on alternative instruments see section 3 5 4 using the above GE Healthcare software packages Please contact your local GE Healthcare representative for availability 3 5 4 IN Cell Image Converter 123 The IN Cell Image Converter 123 see Chapter 10 software converts image files from the following systems for use In the IN Cell Analyzer 3000 e IN Cell Analyzer 1000 e Atto Bioscience Pathway HT e Cellomics ArrayScan VTI HCS Reader e MIAS 2 Multimode Microscopy Reader The conversion produces a run file that can be analyzed by the IN Cell Analyzer 3000 software using the offline analysis mode 25 9003 97UM Rev A 2005 Chapter 3 12 4 Safety warnings handling and precautions 4 1 Safety warning Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Handle as a potentially biohazardous material CAUTION Contains genetically modified material Genetically modified cells supplied in this package are for use in a suitably equipped laboratory environment Users within the jurisdiction of the European Union are bound by the provisions of European Directive 98 81 EC that amends Directive 90 219 EEC on Contained Use of Genetically Modified Micro Organisms These requirements are translated int
67. should perform appropriate control experiments if alternative solubilising agents or concentrations in excess of 196 DMSO are required The cell line has the characteristics detailed in Table 7 3 25 9003 97UM Rev A 2005 Chapter 7 Property Value Measurement method Assay Roscovitine ECs Quality Control stability 37UM 96 G 5uM Assay Nocodazole ECso 165nM 96 G 36nM 20 passages after dispatch Cell Line EGFP expression levels FACS Caliber stability comparable over 20 passages after dispatch when cultured as recommended in Chapter 5 Viability gt 80 CASY1 Cell from frozen Counter and Analyzer System Model TT Table 7 3 Quality control information for cell cycle position reporting cell line Quality control assays were performed by a single operator three repeat assays per cell passage number three cell passage numbers tested P7 P18 and P28 8 replicates per dose Data are mean SD SD shown are standard deviation of the assaus A summary of typical G1S CCPM assay data using Roscovitine and Nocodazole synchronization is shown in Table 7 4 and Table 7 5 In particular Table 7 4 shows the results obtained from a single assay Table 7 5 shows a summary of the results obtained from assays performed by different operators on different occasions giving an indication of inter assay variation Parameter Assay Data H Assays Replicates Roscovitine ECs 33UM 96 G4 1 8 Nocodazole EC 9 196nM G 1 8
68. sponse Figure 5 9 shows a Roscovitine dose response curve for a fixed cell G1S CCPM assay Cells were incubated in the presence of Roscovitine for 24 hours prior to fixation and imaging on the IN Cell Analyzer 3000 Analysis of the percentage of cells with G phenotype was performed using the G1S Cell Cycle Trafficking Analysis Module and provided an EC of 33uM This high EC 9 seems to confirm that the low cellular permeability of Roscovitine impedes efficacy 25 9003 97UM RevA 2005 Chapter 5 23 N C ratio G1 Figure 5 9 Fixed cell assay Roscovitine 1004 Cell number 600 dose response curve 24 hours for U 2 G2 OS cells exhibiting stable expression of G1S CCPM EC of 33uM Roscovitine 9o of cells in G1 Mean SD n 8 replicates per dose R 0 92 Jequunu 92 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 Roscovitine M At concentrations between 5 and 15uM Roscovitine produced a significant increase in the percentage of cells with a G phenotype and a reciprocal effect on G496 can be seen in Figure 5 9 Cell cycle arrest in G at low concentrations and Gi at higher concentrations would seem to reflect the ECz values for Roscovitine against purified Cdk1 cyclin B 0 45u M and Cdk2 cyclin E 0 7uM respectively 5 5 2 G1S CCPM fixed cell assay Nocodazole dose response Figure 5 10 shows a Nocodazole dose response curve for a fixed cell G1S CCPM assay Cells were incubated in the presence of Nocodazole for 16 h
69. t chosen incorrectly Flat field correction not applied or flat field solution intensity too low or saturating 34 Remedy Seed cells at recommended densitu Mitotic apoptotic dead and rounded cells have a smaller surface area in contact with the plate and can therefore become dislodged and lost during wash steps Start fresh batch of cells from an earlier passage number Cells should be expanded and additional vials should be frozen down from the vials delivered with the assay Verify density of cell plating adjust plating density to values that yield optimal assay response Check that the primary parameters are correct and suitable for the cells currently in use Ensure that proper incubation is maintained as consistently as possible during the assay Repeat assay with fresh reagents Use actively growing cells maintained at 37 C 5 CO 95 humidity Pre warm reagents to 37 C Under fixation and therefore loss of fluorescent protein fix as recommended in the protocol Fixed as recommended in the protocol Gentle wash steps without dehydration of cells Check excitation emission and adjust Nuclear stain concentration to recommended level Adjust Nuclear stain incubation time to recommended length Ensure correct plate type selected for correction collar and AF laser power settings Perform Z stack on cells Change AF Offset Apply flat field correction or adjust flat field solut
70. tein and MDM resulted in a cellular population with predominantly nuclear distribution of the sensor whilst cells that had been arrested in G phase using Taxol Nocodazole Colcemide Colchicine and siRNAs against PLK exhibited predominantly cytoplasmic distribution 25 9003 97UM RevA 2005 Chapter 5 29 Control siRNA cyclin E SiRNA PLK sIRNA MCM4 nocodazole olomoucine colcemid The user should be aware that the pleiotropic effects of certain agents can be specific to the cell type method incubation time and concentration used For example U 2 OS cells are P53 and Rb competent but do not always respond to the G M checkpoint especially when using lower concentrations of drugs Therefore the production of a dose response curve is advisable A population of U 2 OS cells arrested predominantly in G phase with irregular large nuclei or dual nuclei can be commonplace when using low concentrations or increased incubation times for Taxol Nocodazole or other agents that cause spindle disruption Time lapse microscopy indicates that this effect is a consequence of delay at the M phase checkpoint but eventual breakthrough and a lack of cytokinesis result in 4n arrest in G These effects are generally not seen at higher concentrations since cells arrest either in G or at the M phase checkpoint However the loss of poorly adhered cells in fixed cell assays will alter results significantly and can cause mis interpretation of drug effects Cells
71. the endogenous Cdk4 inhibitor p16 NK4 and 25 9003 97UM RevA 2005 Chapter 3 11 p14 FF 11 It has been shown that p53 and Rb are required to sustain G4 and G phase arrest induced by DNA damage in tumour cells 12 13 and these genes are crucial determinants of cellular sensitivity to chemotherapeutic agents However the lack of p16 NK and p14 RF expression in U 2 OS cells result in reduced susceptibility to Cdk4 inhibition and the possibility of responses to chemotherapeutic agents that are incomparable with cell lines of differing genotype e g SA0S2 p53 and Rb negative p16 and p14 competent Changes in the level of a single component of the cell cycle machinery can disrupt the complex interplay between Cdks inhibitors and cyclins throughout the cell cycle Therefore it is inappropriate to assume that the pleiotropic effects of a drug upon one cell line can be duplicated in another 3 3 2 Production of clonal U 2 OS cells exhibiting stable expression of the G1S CCPM U 2 OS cells were transfected with the pCORON1002 EGFP C1 PSLD vector using FUGENE 6 Transfection Reagent Roche Cells were grown in complete medium for 48 hours and then in the presence of Geneticin G418 antibiotic at img ml t for approximately four weeks during clonal selection Isolated primary clones 40 were analysed by flow cytometry to confirm the level and homogeneity of expression of the sensor and where appropriate secondary clones were developed and
72. ts for individual cells and the total population include cell number classification cell rounding nuclear area nuclear cytoplasmic and cellular intensity values for the blue green and red channel 5 3 2 IN Cell Analyzer 1000 The U 2 OS cell line exhibiting stable expression of the G1S CCPM can be imaged on the IN Cell Analyzer 1000 instrument and other non confocal and non laser based epifluorescent microscopes The IN Cell Analyzer 1000 produces similar images to those obtained on the IN Cell Analyzer 3000 Figure 5 6 A C Images from the IN Cell Analyzer 1000 can be analysed by the G1S Cell Cycle Trafficking Analysis Module via the simple to use IN Cell Image Converter 123 see Section 3 5 Dose response curves for Roscovitine and Nocodazole generated using this technique are presented in Section 5 5 5 3 25 9003 97UM Rev A 2005 Chapter 5 21 Figure 5 5 Fixed U 2 OS cells exhibiting stable expression of the G1S CCPM imaged on the IN Cell Analyzer 3000 section 5 2 2 A control cells B 24 hours incubation with Roscovitine 250uM and C 16 hours incubation with Nocodazole 10M Images shown are a 1 5th section of the original image Hoechst nuclear marker and cell cycle phase classification as determined with G1S Cell Cycle Trafficking Analysis Module are shown Figure 5 6 Fixed U 2 OS cells exhibiting stable expression of the G1S CCPM imaged on the IN Cell Analyzer 1000 A control cells B 2
73. tsmouth UK and an ORCA ER 12 bit CCD camera Hamamatsu Reading UK Illumination was controlled by means of a shutter in front of the transmission lamp and an x y positioning stage with separate z focus Prior Scientific Cambridge UK controlled multi field acquisition Image capture was controlled by AQM 2000 Kinetic Imaging Ltd All images were collected with a 40x 0 75NA air apochromat objective lens providing a field size of 125um x 125um Following collection of the images the average intensity of an area of the nucleus and cytoplasm was determined at each time point for an individual cell using the Lucida software package Kinetic Imaging The movement of the cell and field was compensated for during the time course Images generated with the Axiovert 100 are comparable with those from the IN Cell Analyzer 3000 see Figure 5 4 After mitosis and cell division lasting approximately 1 hour there is a phase of 4 5 hours where the sensor exhibits predominantly nuclear distribution Rapid changes in sub cellular distribution associated with this phase are indicative of high Cdk2 cuclin E activity The sensor then demonstrates a phase of progressive export from the nucleus over the following 10 hours and a phase of 3 hours when it is exclusively cytoplasmic The length of each of these phases correlates with the reported lengths of M G4 S and G phases for rapidly dividing U 2 OS cells om E kaon 19h 20m Figure 5 7 Rep
74. type of the G1S CCPM sensor can also provide additional data for the classification of cells with respect to progress through the cell cycle see Section 5 3 1 4 2n An 9 1 404 c S M 1 35 D AA EN M 0 cuu 0 095 5125 i M 1 20 j EM fee DOR DE Aw Oo eX Ro iar ae Ope rs o 1 15 e 8 oat AT ete FEE to gt T m 1 10 E Mw Ar J E UN eee i 2105 ARAS T TI LU 1 00 Ott E ESO S ELO Jar Je S Du ee vue ie TU E G 4 7 4 8 4 9 5 0 5 1 5 2 5 3 Log Integrated Nuclear Hoechst Intensity 25 9003 97UM Rev A 2005 Chapter 5 31 Figure 5 22 Dual parameter analysis of fixed asynchronous U 2 OS cells exhibiting stable expression of the G1S CCPM sensor Graph shows an object plot of individual cells after 48 hours growth fixed and imaged on the IN Cell Analyzer 1000 prior to analysis with the IN Cell Analyzer 1000 Morphology Analysis Module GE Healthcare The sub celllular distribution of the G1S CCPM sensor nuclear cytoplasmic ratio of green signal is indicated on the y axis The integrated blue fluorescence due to Hoechst 33342 staining per nucleus is presented on the x axis and provides an indication of DNA content per nucleus Cells with a low integrated hoechst intensity indicative of 2n and high N C ratio are in G G Cells exhibiting DNA replication from 2n to 4n are clearly visible S phase as are 4n cells in G and 4n cells in mitosis indication of phase is provided 6
75. y cytoplasmic After mitosis and division around 25 hours lasting 1 hour daughter cells re enter the cell cycle The upper two panels show the effect of inhibitor compound A Treated cells have a similar parental cell cycle to control cells and progress through a temporally uninhibited mitosis However treated cells do not undergo correct kinetochore attachment and cytokinesis resulting in a large single 4n daughter cell that has arrested in G phase with a fragmented nucleus 5 3 1 4 Analysis using the IN Cell Analyzer 3000 On the IN Cell Analyzer 3000 the degree of translocation of the cell cycle phase marker fusion protein can be determined using the G1S Cell Cycle Trafficking Analysis Module A detailed description of this module can be found in the analysis module user manual The G1S Cell Cycle Trafficking Analysis Module is designed to provide the user with a flexible system for the classification of cells into 4 or fewer phases of the cell cycle The cell cycle status of individual cells can be determined by measurement of G1S CCPM fluorescence intensity sub cellular distribution see Figure 1 4 and Figure 1 5 for phenotypes and classification system used in following sections to determine EC values and other user defined cellular characteristics with one or two additional fluorescent probes The system is designed to provide an easy to use interactive Figure 5 5 decision based method of classification Image analysis outpu
76. your local GE Healthcare representative for recommended microplate details 5 3 1 1 Fixed cell imaging of G1S CCPM on the IN Cell Analyzer 3000 Fixed cell endpoint images of an asynchronous control population see section 5 2 2 for protocol and cells treated with Roscovitine and Nocodazole are shown in Figure 5 2 Treatment of the G1S CCPM stable cell line with a relatively high concentration of Roscovitine a purine derivatised dual Cdk 1 2 inhibitor resulted in a population with predominantly nuclear distribution of the G1S CCPM sensor indicative of Cdk2 inhibition and arrest in G4 Treatment of the G1S CCPM cell line with Nocodazole an inhibitor of microtubule assembly resulted in a significant increase in the percentage of cells with a G phenotype few M phase cells were evident with current fixed cell assay protocol 25 9003 97UM Rev A 2005 Chapter 5 19 5 3 1 2 Live cell end point imaging of G1S CCPM on the IN Cell Analyzer 3000 The G1S CCPM assay can be performed in live cell format as discussed in sections 5 2 1 and 5 2 3 An example live cell G1S CCPM assay performed on the IN Cell Analyzer 3000 is shown in Figure 5 3 The images clearly show that M phase and other phenotypes with low adherence cells are retained using this method and highlight the differences when compared to a fixed cell assay Figure 5 2 5 3 1 3 Live cell kinetic imaging of G1S CCPM on IN Cell Analyzer 3000 Kinetic time lapse images of asynch

G1S Cell Cycle Phase Marker Assay—User Manual

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G1S Cell Cycle Phase Marker Assay&mdash;User Manual

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G1S Cell Cycle Phase Marker Assay—User Manual