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25-8010-50UM external cover - GE Healthcare Life Sciences

1. 1 9 17 25 33 141 49 57 65 73 Colc Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Control Control 2 10 18 26 34 42 50 58 66 74 C Colc Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Mimo Control 3 11 19 27 35 43 51 59 67 75 Control Mimo Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Mimo Control 4 12 20 28 36 44 52 60 68 76 Control E Mimo Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Mimo Control 5 13 21 29 37 45 53 61 69 77 Control F Mimo Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Colc Control 6 14 22 30 38 46 54 62 70 78 Control G Control Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Colc 7 15 23 31 39 47 55 63 71 79 Control B Control Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Colc 8 16 24 32 40 48 56 64 72 80 Control As explained in the IN Cell Analyzer 3000 user manual each run must contain a flat field FF well to compensate for variations in fluorescence intensity across each image It is possible to prepare a microplate solely for this purpose Alternatively a designated well on each plate can contain FF solution When seeding the plate this well must not contain any cells if the auxiliary flat field correction tool is to be applied in the analysis module The plate map shown does not contain a FF well Users should modify their plate accordingly for example by replacing a compound well with the FF well Col
2. Qa e manual 25 8010 50 25 8010 50 up ea 25 8010 51 25 8010 52 25 8010 52 25 8010 53 25 8010 53 G2M Cell Cycle Phase Marker Assay Aessy J9XIe NA ISEUd 93 29 MBI IN CS 7l Amersham L V Biosciences um 25 8010 50UM um 25 8010 50UM Amersham Rev A 2003 EO e Y Biosciences Qua 25 8010 50 25 8010 51 25 8010 52 25 8010 53 G2M Cell Cycle Phase Marker Assay Amersham e Biosciences um 25 8010 50UM Rev A 2003 Front cover A field of U 2 OS cells expressing the G2M cell cycle phase marker with a single cell undergoing cell division As the cell divides the green fluorescent reporter initially moves from the cytoplasm to the nucleus as the cell enters prophase and then the fluorescence diminishes as the cell completes metaphase um 25 8010 50UM Page finder Rev A 2003 Page finder Chapter 1 Introduction 1 1 1 2 LaL 1 2 2 1 2 ox 1 2 4 1 3 The Cell Cycle Cell Cycle Phase Markers llle Cyclin B1 function Cyclin B1 promoter Cyclin B1 cytoplasmic retention sequence CRS Cyclin B1 degradation box D box 0 0 0 0 eee Applications in drug discovery 00000 ee eee eens Chapter 2 Licensing Considerations 2 1 EC ora Chapter 3 Product Contents 3 1 3 2 3 3 fe to tls Bio 3 4 3 5 Components summary GFP expression vector pCORON4004 C
3. Chapter 8 Troubleshooting Guide 8 1 Troubleshooting guide leeren Chapter 9 References 9 1 References lle hn hn Chapter 10 Related Products 10 1 Related products 229 oro REA ECERXER TAX EX E E39 EUER Chapter 11 Appendix 11 1 Restriction map of pCORON4004 CCEGFP um 25 8010 50UM Page finder Rev A 2003 Fig 1 1 The cell cycle Cells that are not actively dividing are in GO phase When a signal is received to divide the cell enters G1 phase During G1 the cell becomes larger and prepares for DNA replication In S phase a second copy of the genome is produced thus doubling the amount of DNA within the cell Once DNA replication is complete the cell enters G2 phase where proteins required for mitosis are synthesized and final checks on the integrity of the DNA are made In mitosis M phase the cell divides to produce two daughter cells each inheriting a copy of the entire genome um 25 8010 50UM Chapter 1 Rev A 2003 Chapter 1 Introduction 1 1 The Cell Cycle The cell cycle Figure 1 1 is the process by which cells replicate their DNA and divide and is therefore one of the most fundamental processes occurring in eukaryotic cells 1 2 Literally a matter of life and death 2 the cell cycle is studied by scientists in a broad range of disciplines who are interested in understanding the mechanisms of this exquisitely regulated process or in
4. Dpnll Dral Drall Dralll Drdl Dsal DsaV Eael Eagl Eam1105l Earl EcIXI ECODZI EcoNI EcoO109l1 EcoRI EcoRII Esp3l Espl Fnu4Hl FnuDIl Fokl Fspl Haell Haelll Hgal HgiAl of cuts al w CO Oo 99 29 12 35 Positions c indicates the complementary strand 133 428 861 889 1385 1535 15 1 1915 1919 1955 2509 3277 3355 5456 3445 3523 3900 4399 4435 4452 4710 4756 4774 5115 5220 5232 5310 5318 5329 5404 5982 231 18 3 4504 5196 5215 301 4102 2231 2219 2949 3133 3999 58685 2023 2819 35368 2 2 77 139 353 391 483 913 929 1054 1166 1241 1295 1567 1622 1623 1639 1640 1922 2624 2679 2696 3108 3268 3491 4008 4043 4544 4895 5591 5809 5822 5943 75 957 1346 1646 3012 3186 3577 3604 3829 4689 1646 3012 5082 257 355 c 1936 c 3450 c 3660 c 4283 c 1646 3012 563 c 1039 1083 c 1282 3252 3684 4416 5428 c 802 301 4102 15 72 77 139 353 391 929 1054 1166 1241 1295 2624 2679 2696 3491 5809 5822 5943 4045 4087 c 343 2 30 33 166 169 265 268 348 351 470 1045 1104 1126 1410 1417 1468 1471 1565 1646 1649 1797 1979 2011 2025 2047 2518 2860 3015 3067 3078 3168 3173 3210 3251 3338 3341 3344 3580 3676 3717 3731 3832 3942 4051 4338 4567 4662 4689 5028 5356 5562 5565 5630 5773 5928 26 130 132 452 1214 1532 2000 2024 2044 2420 2507 3172 3473 3505 3906 3986 4089 4091 4191 4523 5016 5346 5927 387 788 856 913 c 1279 c 2763 c 3430 3455 4000 c 4643 4930 5111 1977 2516 3208 4859 382 207
5. 3 An example of the population distribution of cells in various phases of the cell cycle as determined by the Cell Cycle Trafficking Analysis algorithm is shown in Fig 5 8 Fig 5 8 Population distribution of control cells in various phases of the cell cycle analyzed by the Cell Cycle Trafficking Analysis Module IN Cell Analyzer 3000 assay Fig 5 9 Cell cycle position reporting cell line imaged on the IN Cell Analyzer 1000 Cells were treated as described in Figure 5 4 but were washed in PBS prior to imaging um 25 8010 50UM Chapter 5 Rev A 2003 100 1 GO G1 S phase cells uj G2 phase cells EE Prophase cells EE Mitotic cells 75 50 of cells 25 control 5 3 2 IN Cell Analyzer 1000 It is also possible to use the G2M cell cycle phase marker assay on the IN Cell Analyzer 1000 instrument and other non confocal and non laser based epifluorescent microscopes Figure 5 9 An analysis module for images acquired on IN Cell Analyzer 1000 is under development Control Mimosine Colchicine Fig 5 10 The average fluorescence intensity of a G2M CCPM expressing U 2 OS cell followed over 60 hours During this time the cell undergoes three mitotic divisions An increase in fluorescence is observed as the cell progresses through the cell cycle and a rapid reduction is seen immediately following each round of cell division um 25 8007 50UM Chapter 5 Rev A 2003 5 4 Cell cycle position r
6. 3 Incubate the microplates at 37 C 5 CO2 for the desired period of time and image at defined time points Temporal effects of cell cycle inhibiting drugs can be evaluated visually in the resulting time lapse movies of each well For this assay it is important to use high performance microimaging Fig 5 3 Cell cycle phase reporting U 2 OS cells imaged during a typical assay run performed on the IN Cell Analyzer 3000 Frames shown are a fraction 1 75 th of the entire images captured um 25 8010 50UM Chapter 5 Rev A 2003 equipment capable of returning to the same position in a well reliably Care should be taken to optimize the excitation conditions to minimize detrimental effects on the cell e g DNA damage by UV light and to prevent photo bleaching of the cells 5 3 IN Cell Analysis System The cell cycle position reporting assay has 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 this assay as part of this system Is included in the user manuals for the IN Cell Analyzer 1000 the IN Cell Analyzer 3000 and the Cell Cycle Trafficking Analysis Module 9 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 96 well Viewplate If planning to perform the assay in 384 format please contact
7. 1 2 pCORON4004 CCEGFP expression vector The pCORON4004 CCEGFP vector is supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 0 at 250 pg ml The vector should have the characteristics outlined in Table 7 2 Property Value Limits Measurement method Concentration 250 ug ml UV Absorbance 260 nm in water Purity Minimal A 60 A289 ratio Between UV Vis contamination of the 1 8 2 2 Absorbance DNA construct by RNA 260 nm and or protein 280 nm Expected restriction The restriction Agarose gel Table 7 3 Expected restriction pattern for the pCORON4004 CCEGFP expression vector um 25 8010 50UM Chapter 7 Rev A 2003 pattern Enzyme s EcoRI BamHI Sall Pstl Kpnl Hindlll digests should give electrophoresis fragments of the sizes shown in of cuts NR C0 We NM F amp F Table 7 3 Fragment s size bp 5991 3011 2980 599 3377 23 0 244 4059 999 933 3430 2561 Problem Low assay response positive vs negative controls Low nuclear intensity Image is out of focus IN Cell Analyzer 3000 only o Cells do not adhere to well bottom in plate um 25 8010 50UM Chapter 8 Rev A 2003 Chapter 8 Troubleshooting guide 8 1 Troubleshooting guide Possible causes and remedies Possible cause 1 1 Passage number too high 1 2 Cell density too low or too high 1 3 Incorrect selection of analysis parameters 1 4 Incorrect assay incubation conditions 1 5 Reagent
8. 158 80 00 o Prophase cells Cell cycle r 0 GI 605 9157 5 79 132 132 5b Cell cycle r OHI 577 9081 6 24 121173 70 00 OMitotic cells 54 Cell cycler 0 Az 55 Cell cycle r 0 B2 56 Cell cycle r 0 C2 57 Cell cycle r 0 D2 Cell cycle r 0 E2 Cell cycle r 0 F2 60 Cell cycler 0 G2 616 88 64 7A 244 146 797 33 22 452 113 113 707 8854 8 49 1 56 141 TW 31 35 6 56 1 53 0 56 762 3134 7 22 0 92 0 52 736 88 86 8 97 0 82 136 532 83 16 8 09 1 59 116 60 00 50 00 40 00 30 00 20 00 of cells 61 Cell cycler 0 H2 608 9342 4 61 0 66 132 62 Cell cycler 0 A3 534 8858 7 68 2 43 131 10 00 63 Cell cycler 0 B3 755 8954 6 03 281 146 0 00 amp amp Cell cycle r 0 c3 Cell cycle r 0 D3 Cell cycle r 0 E3 Cell cycle r 0F3 68 Cell cycler 0 G3 68 Cell cycle r 0 H3 Cell cycle r 0 A4 71 Cell cycle r 0 B4 537 8 83 8 75 112 13 739 89 04 8 8 0 35 122 680 88 53 8 09 176 162 75 89 83 7 53 172 0 32 666 3174 4 95 15 18 677 3114 4 58 34 0 83 624 30 71 465 2 56 2 08 738 89 84 6 31 13 136 Control Mimosine Colchicine Cell cycle r 0 C4 631 3048 745 073 127 Cell cycle r 0 D4 631 89 22 7 61 19 127 Cell cycler 0 E4 640 8891 8 53 188 0 63 75 Cell cycle r 0 F4 602 90 86 781 116 0 17 76 Cell cycle r 0 G4 730 9068 6 58 2 05 0 68 Cell cycle r 0 H4 582 9416 3 09 1 03 172 78 Cell cycle ri 70 23 24 53 0 51 4 68 fal jew BR G aoshe NOOR 4 o 4 A S SO
9. 3173 3210 3251 3338 3341 3344 3580 3676 3717 3731 3832 3942 4051 4338 4567 4662 4689 5028 5356 5562 5565 5630 5773 5928 3105 634 1633 2566 257 355 c 1936 c 3450 c 3660 c 4283 c 92 242 1724 2075 2913 2967 4889 5224 5477 5987 64 950 1163 1334 2117 2227 2270 2282 3222 3409 4160 4480 4853 5269 387 520 1068 1557 1782 2038 2050 3226 3532 4033 4421 4609 4762 4820 5151 5434 5550 5613 1914 3899 133 428 861 889 1385 1533 1571 1915 1919 1955 2509 3277 3355 3436 3445 3523 3900 4399 4435 4452 4710 4756 4774 5115 5220 5232 5310 5318 5329 5404 5982 244 c 372 438 573 721 802 805 862 1128 c 1173 c 1176 c 1371 1953 2089 c 2929 c 3467 3677 375 c 4300 4409 4487 5242 5313 c 5465 c 136 162 1649 1958 3015 4564 4713 5636 1821 77 3188 10 47 c 106 124 205 c 231 c 329 356 c 485 c 486 629 759 894 c 975 c 981 c 1075 1212 c 1224 c 1275 c 1395 c 1657 c 1857 c 1897 1937 c 2201 2541 c 2549 2565 c 2843 c 2849 c 2873 2879 2886 c 2889 c 2901 c 3021 c 3157 c 6514 c 3707 4056 0 4115 4709 4915 6 5062 5143 5543 5793 0 5867 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme Mscl Msel Msll MspA11 Mspl Munl Mval Mvnl Mwol Nael Narl Neil Ncol Ndell NgoMI NIalll NlalV Notl Nsil Nspl NspV PaeR71 PinAl Plel PmaCl Pmll Ppu 10l of cuts 2 2 11 32 17 29 37 15 al 25 24 Pe A N rnm IA Positions c indicates the complementary strand 4 3188 5230
10. Contained Use Regulations 2000 Information to assist users in producing their own risk assessments is provided in section 3 3 1 and 3 3 2 of The Genetically Modified Organisim Contained Use Regulations 2000 http www legislation hmso gov uk si si2000 2000283 1 htm 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 vapor 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 a Suitably equipped laboratory environment and should only be used by responsible persons in authorised areas Care should be taken to prevent ingestion o
11. JBC 269 9 6320 4 31 Henderson B R Eleftheriou A 2000 A comparison of the activity sequence specificity and CRM1 dependence of different nuclear export signals Exp Cell Research 256 1 213 224 32 Freshney R I Cloning and selection of specific cell types in culture of animal cells 3rd edition Wiley Liss Inc chapter 11 ppl161 178 1994 um 25 8010 50UM e Chapter 9 Rev A 2003 um 25 8010 50UM Chapter 10 Rev A 2003 Chapter 10 Related products 10 1 Related products Product Name Code GFP Assays GFP PLC PH domain Assay 25 8007 26 GFP Racl Assay 25 8007 27 GFP MAPKAP k2 Assay 25 8008 82 AKT1 EGFP Assay 25 8010 17 EGFP 2x FYVE Assay 25 8010 21 Use of these products is limited in accordance with the type of license purchased Please contact your local representative for more details IN Cell Analysis System IN Cell Analyzer 3000 25 8010 11 Cell Cycle Trafficking Analysis Module 63 0050 71 IN Cell Analyzer 1000 25 8010 26 e 1 Enzyme Aatl Aatll Acc6 5l Accl Acil Acs Acyl Afll Agel Alul Alw441 Alwl AlwNI Aosl ApaL Apol Asel Asnl Asp 700 Asp 18 AspE AspHI of cuts 2 l 3 1 13 CO M NM NYO HR W B OF No m Chapter 11 Appendix 11 1 Restriction map of pCORON4004 CCEGFP The following enzymes do not cut the vector Acclll AfIIII Apal Ascl Bcll Bglll BseAl BsiWl Bsp120l BspEl Bst1107l BstEll Bsu36l Ecl136ll Eco47lll EcoRV Kspl
12. Mlul Mrol Ndel Nhel Nrul Pacl PfIMI Pmel Sacl Sacll SgrAl SnaBl Spel Sse838 7l Swal Van91l Xbal Positions c indicates the complementary strand 82 2911 4163 630 1629 2562 1636 11 c 26 c 109 169 223 c 267 c 270 c 340 1104 1145 1212 1251 1389 1502 1562 1565 1645 c 1649 1949 2010 c 2024 c 2027 c 2055 2082 2460 c 2486 c 2499 2507 c 2575 c 2760 2772 2781 2793 2803 2814 2860 3015 3078 3172 c 3236 c 3337 c 3340 c 3580 3620 c 3625 3675 c 3691 3717 3773 c 3832 3904 3942 3968 3978 4017 4191 c 4238 4337 c 4446 c 4523 c 4567 4688 c 4734 4925 6 5016 c 5378 538 6 5522 560320 5b753 6 5772 c 5899c 5927 c 15 1757 2411 2422 3106 3808 4160 4542 2960 1603 32 347 369 505 583 859 906 939 1011 1044 1260 1308 1419 1593 1796 2141 2398 2588 2876 2930 3212 36 0 4031 4050 4729 4792 4892 5413 5670 5716 5806 3913 4410 5656 G56 0 684 c 897 136000 1979 191440 19223 2917 32985 325016 3531 SS95 c 3908 4443 4447 c 4764 5227 c 5228 5324 c 5326 5412 5977 c 586 658 682 694 5561 1977 2516 3208 4859 3913 4410 5656 15 1757 2411 2422 407 4907 407 4907 124 4482 630 1629 2562 5082 1495 3219 3409 3917 4414 4499 5660 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand Asp Asull Aval Avall Avill Avrll BamHI Banl Banll BbrPI Bbsl Bbvl Bcgl Bfal Bfrl Bgll BInl Bmyl Bpml Bpu1102l BpuAl BsaAl BsaBl Bs
13. Pvull Rcal Rsal Rsrll Sall Sapl Sau3Al Sau96l Scal ScrFI SexAl SfaNI Sfcl Sfil Sful Smal Snol Sphl SspBl Sspl Stul Styl Taq Tfil Thal of cuts 15 32 22 23 Positions c indicates the complementary strand 301 4480 4853 545 789 3159 136 1958 4713 32 2588 9212 4137 4242 5250 632 1315 1597 1631 2564 3412 3925 4601 3622 1635 257 3450 c 3660 c 133 428 861 889 1385 1533 1571 1915 1919 1955 2509 3277 3355 3436 3445 3523 3900 4399 4435 4452 4710 4756 4774 5115 5220 5232 5310 5318 5329 5404 5982 105 301 316 462 1058 1459 1542 1946 2234 3622 4102 4718 4940 4957 5036 4601 74 79 141 355 393 485 915 931 1056 1168 1243 1297 1569 1624 1625 1641 1642 1924 2626 2681 2698 3110 3270 3493 4010 4045 4546 4897 5593 5811 5824 5945 26 8 834 c 984 c 1262 1277 1376 1755 c 2447 c 2487 2669 2741 3064 c 3319 c 3405 3469 3535 c 3 44 3928 c 4022 4381 c 4630 4821 c 5873 c 541 785 2005 3155 4836 5514 5705 2865 3 88 1625 1642 3913 4410 5656 2000 2732 3911 1595 2436 4277 827 2911 304 893 2523 2819 2912 3538 219118 400 935 1229 1256 1271 1400 1636 1677 1918 2193 2955 37219 3375 3399 3435 3597 3788 3887 4428 5872 5977 21 1615 2934 3591 3725 3884 26 130 132 452 1214 1532 2000 2024 2044 2420 2507 3172 3473 3505 3906 3986 4089 4091 4191 4523 5016 5346 5927 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme of cuts Tru9l 29 Tsp509 15 Tth111l 1 Xhol 1 Xholl Is Xmal
14. UK was used to control multi field acquisition Image capture was controlled by AQM 2000 Kinetic Imaging Ltd All images were collected with a 40x 0 75 NA air apochromat objective lens providing a field size of 125x125 um Following collection of the images the average intensity of an area of the cell was determined at each time point using the Lucida software package Kinetic Imaging The level of reporter protein fluorescence was recorded in the cytoplasm from a region of interest ROI drawn in an area adjacent to the nucleus The movement of the cell was compensated for during the time course The intensity readout was background subtracted and normalized against basal levels of cyclin B1 and plotted against time At mitosis a large increase in average fluorescence is observed The fluorescence is rapidly degraded as the cells enter G1 phase This cycle is repeated each time the cell divides over the 60 hour time course shown in Fig 5 11 2 WY c cD c cD U c e9 U WY D i O va 30 40 time hours Fig 5 12 Flow cytometry on fixed and propidium iodide stained cell cycle reporting cell line a Histogram of red fluorescence b Dot plot of red versus green fluorescence um 25 8007 50UM Chapter 5 Rev A 2003 5 5 Cell cycle position reporting using flow cytometry The G2M CCPM phase marker assay can also be used to report cell cycle using a flow cytometer In Fig 5 11 the reporting cell line
15. Untreated cells Colc Control Cells treated with a compound that inhibits cells in mitosis e g O l1ug ml colchicine Mimo Control Cells treated with a compound that inhibits cells at the G1 S boundary e g 2mM Mimosine Cmpd Test Compound um 25 8010 50UM Chapter 5 Rev A 2003 sensitivity of the assay e The use of intercalating nuclear dyes Hoechst or DRAQ5 is not possible because these dyes inhibit mitosis 5 2 2 Seeding and preparation of cells for a screen of cell cycle inhibiting compounds Seed 3000 cells per well in a 96 well microplate or 0 8 x 103 cells per well in a 384 well microplate Place in a humidified 37 C 5 CO incubator for 24 h as described in section 5 1 5 It is recommended that cells are in log phase growth and are maintained at 37 C during the assay Reagents used during the assay should be pre warmed to 37 C It is essential that the number of cells per well in the assay plates is consistent in order to minimize assay variability The following assay protocol is configured for 96 well microplates The cells should be seeded in the appropriate microplate the day before the experiment Decant the culture medium and replace with Growth medium containing 2mM mimosine O 1 ug ml colchicine test compound or Growth medium only for controls A typical 96 well microplate map containing control and test wells in a recommended configuration is shown in Fig 5 2 Control
16. descirbed in section 5 5 The percentage of cells classified as G2M using the IN Cell analyzer 3000 and Cell Cycle Trafficking Analysis Module was 24 3 for control populations 9 9 for mimosine treated cells and 78 for colchicine treated cells Fig 5 12 These percentages closely agree with results obtained using flow cytometry analysis Fig 5 13 where the corresponding values were 23 6 for control populations 10 4 for mimosine treated cells and 67 8 for colchicine treated cells The results confirm that the compound treatment regimes specified in the G2M CCPM assay protocol are effective and that the image analysis module correctly identifies cells in G2 M phase 100 4 i G0 G1 S phase cells El G2 phase cells 90 4 Prophase cells 80 Bi Mitotic cells 70 4 9 996 78 0 96 xw 60 O 504 6 40 4 30 4 20 4 m Control Mimosine Colchicine Figure 5 13 Flow cytometry analysis Following treatment for 24 h in the absence or presence of cell cycle inhibiting compounds cells were fixed and stained with propidium iodide Flow cytometry was performed as described in section 5 5 Results are consistent with those obtained using the IN Cell Analysis system and software Figure 5 14 Colchicine dose response using the supplied cell cycle phase marker cells The data were collected 24 hours after addition of the drug yielding an EC50 of 32 ng ml Error bars indi
17. incellanalyzer uk amershambiosciences com for microplate details 5 3 1 1 Kinetic assay using the IN Cell Analyzer 3000 Figure 5 3 shows selected frames of the same cell captured on the IN Cell Analyzer 3000 The cells were plated on a Packard Viewplate and imaged every 10 minutes for 8 hours The figure shows a cell that is dividing normally The six frames show a cell that was Initially in G2 and that divides into two daughter cells Time lapse movies showing the cell cycle position reporter in kinetic mode can be viewed by visiting http www amersham com drugscreening Prophase Metaphase Telophase Cytokinesis G1 phase Fig 5 4 End point assay images Cells were seeded onto a Packard Viewplate at 3000 cells per well and incubated at 37 C 5 CO2 for 24 hours before adding 2 mM mimosine or 0 1 ug ml colchicine to the wells After 24 hours the wells were supplemented with 10 uM DRAQ5 10 ul well and following a 37 C incubation for 15 minutes the cells were imaged on an IN Cell Analyzer 3000 Cells in a range of different phases of the cell cycle are observed in the control wells Predominantly mitotic cells are observed in the colchicine treated sample bright rounded and in pairs Evenly stained flattened cells predominante in the mimosine treated sample almost all G1 Fig 5 5 Initial measurements for the cell cycle trafficking module analysis The signal channel nuclear intensity the cytoplasmic ring intensity a
18. 2 Xmalll 2 Xmnl 2 Positions c indicates the complementary strand 230 407 704 1608 1669 1811 1872 2018 2289 2387 2404 2415 2427 2438 2961 3950 4131 4503 4868 4907 5142 5195 5209 5214 5266 6 15 404 821 1757 1821 2411 2422 2448 2666 2738 2830 4649 4904 5210 3224 1 889 1385 2509 3277 3523 3900 4435 4452 5220 5232 5318 5329 5982 1623 1640 1646 3012 124 4482 um 25 8010 50UM Chapter 11 Rev A 2003
19. Dimethylsulfoxide DMSO Sigma D 2650 or equivalent e L Glutamine 200Mm 100 x Gibco catalogue 25030 024 e Mimosine Sigma M 0253 e Demecolcine Colcemid N Deacetyl N methyl colchicine Sigma D1925 10 ml sterile at 10 ug ml e Standard tissue culture plastic ware including tissue culture treated flasks T flasks centrifuge tubes 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 Streptomycin 1 v v L Glutamine and 1 v v Geneticin working concentration 1 mg ml e Cryopreservation medium McCOYS 5A medium modified supplemented with 10 v v FBS 1 v v Penicillin Streptomycin 1 v v L Glutamine and 10 v v DMSO e Nuclear stain DRAQ5 BioStatus Prepare working solution of 10 uM DRAQ5 by diluting a 5 mM stock solution 1 500 in growth medium e Mimosine Prepare a 20 mM stock solution by dissolving 25 mg in 6 25 ml of warmed growth media and place at 37 C for 1 hour Roller mix until dissolved 1 1 5 hours and filter sterilize The solution can be stored at 4 8 C for up to 4 weeks Dilute 1 10 in warm growth media before use e Demecolcine stock at 10 ug ml Dilute 1 100 in warm growth media before use e 4 Formalin solution Sigma HT 50 1 2 5 1 3 Cell thawing procedure Two cryo vials each containing 1 x 106 cells in 1 ml of Cryopreservation medium are
20. be distinguished cells that are in G1 S which are non fluorescent or dimly fluorescent in the cytoplasm cells that are in G2 which are brightly fluorescent in the cytoplasm cells in prophase which are bright all over with nuclei brighter than the cytoplasm cells in the rest of mitosis which are rounded and intensely green in the entire cell um 25 8010 50UM Chapter 1 Rev A 2003 use is in toxicological screening during lead optimization Earlier detection of toxic compounds that disrupt the cell cycle may be expected to reduce drug development costs Multiplexing with a second reporter may be valuable where it is suspected that an agent will exhibit cell cycle dependent efficacy or toxicity Use of the CCPM assay in conjunction with additional fluorescent probes for ligand binding ion flux or other cellular processes will enable the cell cycle dependency of many signalling pathways utilized by drugs or other therapeutic regimes to be investigated Multiplexing the CCPM assay with other dynamic probes allows correlation of cellular events and processes with cell cycle position A number of cell cycle dependent responses to cell stimulation have been reported For example expression of endothelin receptors can be correlated with variation in endothelin induced apoptosis 16 Vasopressin induced calcium mobilization varies with cell cycle dependent expression of different G proteins 17 Cell cycle dependent responses to
21. elucidating targets for therapeutic intervention 3 4 5 6 The complexities of the cell cycle have been the subject of intense and varied study over the past century 7 and are likely to remain so for the foreseeable future The G2M Cell Cycle Phase Marker assay allows researchers and screeners to monitor the cell cycle phase of individual cells in real time In particular the assay has been designed to resolve and quantify cells at the G2 to M transition point The cell based assay therefore has a number of potential applications including screening for anti proliferative compounds that maybe useful in treating cancer and other proliferative disorders and functional screening of orphan targets The assay may also be used in secondary screening and lead optimization studies to detect undesirable toxic side effects of lead compounds earlier in the drug development In profiling studies the assay could be used to investigate the cell cycle dependence of key processes or pathways such as receptor mediated signalling Cell division Replication Fig 1 2 G2M cell cycle phase marker design Location and expression of the fluorescent reporter are under the control of various cell cycle dependent elements Synthesis is controlled by the cyclin B1 promoter which limits production to late S and G2 phases of the cell cycle Destruction of the reporter is controlled by the cyclin Bl D Box which mediates rapid degradation during mitosi
22. in the presence of Geneticin G418 Sigma G 7034 at 1 mg ml for approximately four weeks The cells were then sorted on a high speed FACS into 96 well plates containing conditioned media one fluorescent cell well After 10 days the plates were imaged on the IN Cell Analyzer 3000 to determine which wells contained clonal cells The clones were harvested expanded and analyzed by flow cytometry Clones that met our selection criteria were processed further and one of these lines C 8E6 is supplied The cells are mycoplasma negative details available on request 3 4 Materials and equipment required The following materials and equipment are required but not provided e Microplates For analysis using the IN Cell Analyzer 3000 Packard Black 96 Well ViewPlates Packard Cat 6005182 are recommended For assays in 384 well format please email incellanalyzer uk amersham com for recommendations e A CASY 1 Cell Counter and Analyzer System Model TT Scharfe System GmbH is recommended to ensure accurate cell counting prior to seeding Alternatively a hemocytometer may be used e Environmentally controlled incubator 5 CO 95 relative humidity 37 C e mager microscope e g IN Cell Analyzer 3000 or IN Cell Analyzer 1000 e Controlled freezing rate device providing a controlled freezing rate of 1 C per min eg Nalgene Mr Frosty Sigma C1562 e Standard tissue culture reagents and facilities see also section 5 1 1 3 5
23. protein can be excised using Hindlll and Sall The sequence of the construct is available upon request Please e mail incellanalyzer uk amersham com A detailed restriction map is available in Chapter 11 coRI 15 Cyclin B1 promoter HindIII 367 Ps 545 y Psi 789 fi Ampicillin Resistance Gene BamHI 889 CyclinB1 N terminus EGFP pCORON4004 CCEGFP 2991 bp D Sall 1635 L PolyA Signal 1 F BamHI 3900 Synthetic poly A 1 f1 ori Neomycin Resistance Gene E Ps 3159 SV40 enhancer early promoter HindIII 2928 3 3 Cell cycle reporting U 2 OS derived cell line NIF2033 3 3 1 U 2 OS parental cell line The parental cell line U 2 OS ATCC HTB 96 is a human osteosarcoma cell line derived from the thighbone of a15 year old Caucasian female 21 22 23 Unlike most human carcinoma cell lines U 2 OS is positive for p53 24 um 25 8010 50UM Chapter 3 Rev A 2003 p53 Is required to sustain the G2 phase arrest induced by DNA damage in tumor cells 25 26 Studies have shown that the p53 status of tumor cell lines is a crucial determinant of cellular sensitivity to chemotherapeutic agents because the growth of cell lines with mutant p53 is inhibited less than that of cells expressing wild type protein 27 3 3 2 U 2 0S derived cell cycle reporting cell line U 2 OS cells were transfected with the pCORON4004 CCEGFP vector using the FuGENE 6 Transfection Reagent method Roche Transfected cells were grown
24. receptors and insulin like growth factor Il mannose 6 phosphate IGF II M6P receptors and synthesize IGF II autocrine growth stimulation by IGF II via the IGF I receptor J Cell Physiol 159 531 541 1994 24 Landers JE et a Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild type p53 protein Cancer Res 57 3562 3568 1997 25 Bunz F et al 1998 Requirement for p53 and p21 to sustain G2 arrest after DNA damage Science 282 1497 1501 26 Flatt P M et al 2000 p53 Regulation of G2 checkpoint Is retinoblastoma protein dependent Mol Cell Biol 20 4210 4223 27 O Commor P M et al 1997 Characterisation of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth inhibitory potency of 123 anticancer agents Cancer Research 57 4285 4300 28 Ullman K S Powers MA and Forbes D J 1997 Nuclear export receptors from importin to exportin Cell 90 6 967 970 29 Yang J Bardes E S Moore J D Brennan J Powers M A Kornbluth S 1998 Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1 Genes Dev 12 14 2131 43 30 Nishi K Yoshida M Fujiwara D Nishikawa M Horinouchi S Bepp T 1994 Leptomycin B targets a regulatory cascade of CRM1 a fission yeast nuclear protein involved in control of higher order chromosome structure and gene expression
25. the CCK B gastrin ligand Cl 988 have also been observed 18 Cell cycle position can also significantly alter response to chemo therapeutics and radiation Wortmannin has been shown to preferentially radiosensitize cells in G1 19 and similarly the result of combined taxol and radiation treatment have been shown to vary with the cell cycle 20 G1 S G2 Prophase Mitosis um 25 8010 50UM Chapter 2 Rev A 2003 Chapter 2 Licensing considerations 2 1 Legal Use of this product is limited as stated in the terms and conditions of sale These vary in accordance with the product code purchased Description Product Code G2M Cell Cycle Phase Marker Screening Applications 25 8010 50 G2M Cell Cycle Phase Marker Research Applications 25 8010 51 G2M Cell Cycle Phase Marker 6 month assay evaluation 25 8010 52 G2M Cell Cycle Phase Marker 12 month assay evaluation 25 8010 53 This product is the subject of patent application PCT GBO2 04258 in the name of Amersham Biosciences This product is sold under license from Biolmage A S under patents US 6172188 US 5958713 EP 851874 EP 815257 and under international patent application PCT EPO1 06848 and other pending and foreign patent applications Invitrogen I P Holdings Inc formerly Aurora Biosciences Corporation under US patents 5 625 048 5 777 079 5 804 387 5 968 738 5 994 077 6 054 321 6 066 476 6 077 707 6 090 919 6 124 128 6 319 969 6 403 374 European paten
26. was grown in the presence or absence of either 2 mM mimosine or 100 ng ml colchicine for 24 hours The cells were then trypsinized and counted with a haemocytometer 1 x109 cells were fixed and permeabilized using the cytofix cytoperm kit BD Pharmingen following the manufacturer s instructions The samples were incubated in 5 ug ml propidium iodide 0 4 Triton 50 ug ml RNase for 10 minutes prior to analysis on the FACScalibur flow cytometer Becton Dickenson The left side of the trace shows the histograms of the propidiumiodide red fluorescent channel and confirms that the mimosine and colchicine drug treatment have exerted the expected effects The dot plots on the right hand side of Fig 5 11 show the green reporter fluoresence plotted against the red propidium iodide fluorescence The two fluorescence signals are tightly correlated showing that the GFP based assay can be used as an alternative to flow cytometry analysis a b G1 S G2M CONTROL CELLS Events Green Diagonal pattern confirms that cells with more GFP are in G2 M part of the cell cycle CELLS ae E A The majority of i colchicine treated cells mE have a high green fluorescence Events Green Majority of cells are in G2 M as des COLCHICINE TREATED MIMOSINE TREATED CELLS Events Green Majority of cells are in G1 The majority of mimosine treated cells have a low green fluorescence Figure 5 12 Analysis us
27. 3 2081 3109 5730 77 82 90 106 203 318 463 495 547 959 1059 1348 1460 1648 1947 2236 2378 2528 2853 2859 2868 2911 3014 3188 3579 3606 3831 4104 4691 4958 5038 5496 5930 5948 5959 2006 3816 3992 4550 5280 c 5858 c 1495 3219 3409 3917 4414 4499 5660 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme Hhal HinP1 Hincll Hindll Hindlll Hinfl Hpal Hpall Hphl Ital Kasl Kpnl Ksp632l Mael Maell Maelll Maml Mbol Mboll Merl Mfel MIuNI Mnll of cuts 36 36 12 32 Lo 95 10 14 18 o 24 48 Positions c indicates the complementary strand 45 51 132 381 445 452 1175 1216 1532 1978 2002 2015 2024 2046 2072 2080 2517 3100 3108 3172 3209 3475 3505 3507 3735 3988 4091 4191 4523 4860 4953 5346 5455 5629 5729 5796 43 49 130 379 443 450 1173 1214 1530 1976 2000 2013 2022 2044 2070 2078 2515 3098 3106 3170 3207 3473 3503 3505 3733 3986 4089 4189 4521 4858 4951 5344 5453 5627 5727 5794 1609 1637 1812 1609 1657 19812 367 2928 21 386 1615 2276 2298 2934 3591 3725 3777 3884 5083 5600 1609 1812 8 162 322 485 914 977 1037 1568 1604 1624 1641 1923 2124 3011 3088 3110 3138 3269 3359 3426 3607 4010 4044 4545 4787 4897 4964 4998 5402 5592 5618 57 65 369 381 c 903 c 1233 1257 1386 2228 3284 c 4062 c 4071 c 4355 c 4390 4596 c 5012 5239 2 30 33 166 169 265 268 348 351 470 1045 1104 1126 1410 1417 1468 1471 1565 1646 1649 1797 1979 2011 2025 2047 2518 2860 3015 3067 3078 3168
28. 407 704 1608 1669 1811 1872 2018 2289 2387 2404 2415 2427 2438 2961 3950 4131 4503 4868 4907 5142 5195 5209 5214 5266 774 916 1066 1243 1372 3543 3825 3864 4311 4670 4829 32 342 2588 3212 3980 4446 5387 5632 87 162 322 485 914 977 1037 1568 1604 1624 1641 1923 2124 3011 3088 3110 3138 3269 3359 3426 3607 4010 4044 4545 4787 4897 4964 4998 5402 5592 5618 5765 1821 74 79 141 355 393 931 1056 1168 1243 1297 2626 2681 2698 3493 5811 5824 5945 26 130 132 452 1214 1532 2000 2024 2044 2420 2507 3172 3473 3505 3906 3986 4089 4091 4191 4523 5016 5346 5927 32 155 247 315 449 469 977 1037 1050 1094 1103 1957 1987 2019 2021 2063 2090 2120 2657 2729 2780 2859 2865 3097 3181 3204 3343 3349 3466 3502 3549 3816 3912 4964 5352 5924 5972 2125 3608 31006 485 915 1569 1624 1625 1641 1642 1924 3110 3270 4010 4045 4546 4897 5593 2929 2819 3538 135 428 861 889 1385 1555 1571 1915 1919 1955 2509 3277 3355 5456 3445 3523 3900 4399 4435 4452 4710 4756 4774 5115 5220 5232 5310 5318 5329 5404 5982 2123 3606 438 1122 1152 1347 1542 1587 1689 2527 2660 2732 2823 2980 3325 3511 3542 3568 4057 4141 4246 4639 4675 4753 4763 5254 5974 107 464 632 644 891 923 1461 1631 2156 2168 2189 2564 2630 2702 3107 3142 3902 4195 4785 4996 5037 5131 5903 5942 1646 2662 2734 2660 2732 3511 4057 3788 1 1603 394 2284 2292 c 3771 c 5091 5594 c 65 65 2658 2730 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme PpuMI Psp14061 Pst Pvul
29. CEGFP NIF2034 Cell cycle reporting U 2 OS derived cell line NIF2033 U 2 OS parental cell line lle U 2 OS derived cell cycle reporting cell line Materials and equipment required leeren Software requirements Chapter 4 Safety Warnings Handling and Precautions 4 1 Safety warnings 4 2 Storage 4 3 Handling d2 l WOCIOl a item wa 225 2 COWS pass e uci amp 5 Chapter 5 Cell Assay Design 9 1 Od ls Suus Subs 5 1 4 945 9 140 Culture and maintenance of U 2 OS derived Cell cycle reporting cell line Tissue culture media and reagents required Reagent preparation Cell thawing procedure Cell sub culturing procBQHll Bs 6 aa uuo tare ewe Dur bw be RE EORR Cell seeding procedure Cell freezing procedure um 25 8010 50UM Page finder Rev A 2003 Od GONE Ma ET O acea xc aca bees ee A 5 2 PASS AV SEE TID cette a gx SER aac dC EE e ee es fa Desi Ge D 2 1 Generi ASSAY OB UD uas a drca x A cn Nc ee er CUR TC dee ah D 2 Lb End pont HOO ai axi ur Rea sete dm MEE UP eR xe 5 2 1 2 Advantages of end point method 0 04 5 2 1 3 Disdvantages of end point method S2 I BURG menog urat acta rer oe eo R V rd edd EE Sd ee ES 5 2 1 5 Advantages of kinetic method llle 5 2 1 6 Disadvantages of kinetic method 008 5 2 2 Seeding and prepar
30. Software requirements IN Cell Analyzer 3000 Images acquired using IN Cell Analyzer 3000 can be analyzed using Cell Cycle Trafficking Analysis Module product code 63 0050 71 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 IN Cell Analyzer 1000 An analysis module for images acquired on IN Cell Analyzer 1000 is under development Please contact your local representive for availability or e mail incellanalyzer amersham com Other confocal or epifluorescence microscopes Suitable software will be required for acquisition and analysis of images on these microscopes um 25 8010 50UM Chapter 4 Rev A 2003 Chapter 4 Safety warnings handling and precautions 4 1 Safety warnings 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 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 which amends Directive 90 219 EEC on Contained Use of Genetically Modified Micro Organisms These requirements are translated into local law which MUST be followed In the case of the UK this is The GMO
31. a representative experiment exported to Microsoft Excel 25 8010 50UM Chapter 5 Rev A 2003 Once the nuclear cytoplasmic ratio is determined it is used to calculate the modulus of one minus the ratio As shown in Figure 5 6 four different phases in the cell cycle can be determined by analyzing Inuc and a 1 ratiol GI S G2 Prophase Mitosis Inuc very low Inuc very low Inuc high Inuc very high Icyt Low Icyt High Icyt high Icyt high Nuc Cyt low Nuc Cyt very low Nuc Cyt high Nuc Cyt very high 1 ratio low 1 ratio high 1 ratio low 1 ratio high Example numbers Inuc 93 Inuc 261 Inuc 435 Inuc 739 Icyt 108 Icyt 669 Icyt 366 Icyt 295 Nuc Cyt 0 87 Nuc Cyt 0 39 Nuc Cyt 1 19 Nuc Cyt 2 5 1 ratiol 0 13 1 ratio 0 61 1 ratiol 0 19 1 ratiol 1 5 Following image analysis the population data are exported into a comma delimited text file which can be imported into Microsoft Excel Figure 5 8 S Ele Edit view Insert Format Tools Data Window Hep w T al Cycle Well Msg Mpast G0 Gl G2 PPhse Mitosis Percent of Cells in different cell phases Cell cycle r 0 Al 574 3123 4 38 174 2 09 Cell cycle r 0 Bi 557 9048 4 95 251 245 48 Cell cycle r 0 C1 650 87 85 8 62 2 15 138 100 00 E G1 cells 48 Cellcucler oo 622 873 10 61 1 29 08 50 Celleucler 0 EI 636 8952 786 Moo 0 90 00 m G2 cells Cell cycle r 0 F1 570 8877 7 54 21
32. aHl Bsal BsaJl BsaWl Bsgl BsiEl BsiHKAI BsiYl Bsll 1 A 0 RA me N O N m 30 3224 3788 1 36 1623 1640 301 1542 3622 4718 4940 1977 2516 3208 4859 2912 889 3900 630 642 921 1629 2187 2562 3105 3140 5129 2157 3471 65 800 19 c 41 177 251 c 334 c 337 c 456 c 103 1 c 1137 1421 1428 1454 c 1457 c 1783 c 1990 2058 2529 3053 c 3179 3221 3237 c 3330 c 3742 4037 c 4648 c 5039 5342 c 5548 c 5551 c 5641 1010 4544 c 92 242 1724 2075 2913 2967 4889 5224 5477 5987 2960 1987 2865 4964 2912 783 926 1055 1304 1495 2157 3052 3145 3219 3409 3471 3917 4414 4499 5660 1930 1570 5013 343 800 65 2228 3410 1914 3899 3106 3808 4160 4542 5016 139 304 893 914 1054 1217 1241 1296 1623 1624 1640 2523 2624 2696 2819 2854 2863 2912 3269 3538 5810 321 1603 3137 4786 5617 5764 1014 c 1111 1435 136 162 1649 1958 3015 4564 4713 5636 1495 3219 3409 3917 4414 4499 5660 102 175 199 491 804 1055 1218 1568 2009 2335 2820 3087 3631 4044 5492 5771 59957 5955 102 175 199 491 804 1055 1218 1568 2009 2335 2820 3087 3631 4044 5492 o 7 1 5957 5955 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme BsmAl BsmFl Bsml Bsp1286l BspD BspHI BspMI BspWI BsrBI BsrDI BsrF I BsrGI Bsrl BssHI BstBl BstNI BstUl BstX BstYl Celll Cfol Cfr10l Clal Csp45l Csp6l Ddel Dpnl of cuts D N Ww A 17 17 29 13 36 MOON 19 3d Positions c indicate
33. ation of cells for a screen of cell cycle inhibiting COMPOUNCS ssa rom nmn n a as 5 2 3 End point screen for cell cycle perturbing drugs 5 2 4 Fixed cell screen for cell cycle perturbing drugs 5 2 5 Kinetic assay for cell cycle perturbing drugs 5 3 IN Cell Analysis System 0020 e eee eee ee ees DSL IN CEAN S000 uu scuce ibas e CIRC E 5 3 1 1 Kinetic assay using the IN Cell Analyzer 3000 5 3 1 2 End point assay using the IN Cell Analyzer 3000 5 3 1 3 Analysis using the IN Cell Analyzer 3000 Do INXeSILADalyzer EODD s 2n4262 xr pECACE RR X E S Fra daw baw Y 5 4 Cell cycle position reporting on epifluorescence microscopes 5 5 Cell cycle position reporting using flow cytometry 5 6 Assay characterization eee ee es 5 6 1 Gell cycle validation 1066 os dew ee ed Ros 5 6 2 Colchicine dose response sica kk Seed Se rack ok CR a Bawa 5 6 3 Leptomycin inhibition of nuclear export eee Chapter 6 Vector use details 6 1 General guidelines for vector use 2 1 2 2 00 eee es 6 2 Transfection with pCORON4004 CCEGFP 6 2 1 FuGENE 6 Transfection Reagent protocol 6 3 Stable cell line generation with pCORON4004 CCEGFP Chapter 7 Quality Control 71 Cell cycle position reporting cell line 7 2 pCORON4004 CCEGFP expression vector
34. ay requires CDC34 and initiates DNA replication by degrading a CDK inhibitor The second pathway involves a large protein complex called the anaphase promoting complex APC or cyclosome that initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic Fig 1 3 Cyclin B1 and the cell cycle During Gl lower right corner cyclin B1 is absent during S phase lower left corner cyclin B1 is produced and begins to appear in the cytoplasm of the cell where it binds to CDC2 During G2 upper right corner the cyclin B1 CDC2 complex shuttles between the cytoplasm and the nucleus by a Crm1 dependent pathway Because the rate of nuclear export is much faster than its import the protein is localized predominantly in the cytoplasm At the start of M phase prophase upper right corner nuclear export is inhibited and the cyclin BI CDC2 complex moves to the nucleus As mitosis proceeds cyclin Bl is specifically degraded so that once the cells have re entered Gl phase very little cyclin B1 is present provided with permission from BioCarta www biocarta com um 25 8010 50UM Chapter 1 Rev A 2003 cyclins The N terminal domain of cyclin Bl contains a conserved 9 amino acid motif RTALGDIGN called the destruction box D box that is necessary for cyclin B1 ubiquitination and subsequent degradation Although deletion of the N terminal region does not interfere with the capacity of mitotic cycl
35. c Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd A Control 5 2 3 End point screen 1 Seed 3000 cells per well in 100 ul of Growth medium and incubate for 24 hours at 37 C 5 CO5 um 25 8010 50UM Chapter 5 Rev A 2003 2 Remove media from the plates by inversion and blotting onto a pad of sterile tissues Prepare control wells containing either Growth medium 2 mM mimosine see section 5 1 2 or O 1 ug ml colchicine in Growth medium Dilute the test compounds and dispense onto the cells Each well should contain a final volume of 100 ul 3 Incubate the microplates at 37 C 5 CO for 24 hours In this time the majority of untreated cells would be expected to complete a cell cycle In the presence of a cell cycle perturbing drug the normal ratio of cells in each phase will be altered 4 To assist in object identification it is necessary to stain the cell nuclei prior to imaging Dilute nuclear stain DRAQ5 or Hoechst 33342 to 10 uM and add 10 ul well using a multi channel pipette or automatic dispenser and mix 5 Incubate for 15 20 minutes at 37 C before imaging the plate 9 2 4 Fixed cell screen 1 Seed 3000 cells per well in 100 ul of Growth medium and incubate for 24 hours at 37 C 5 CO 2 Remove media from the plates by inversion and blotting onto a pad of Sterile tissues Prepare control wells containing either Growth medium 2 mM mimosine see section 5 1 2 or O 1 ug ml colchicine in Growth medium Di
36. cate SD n 8 replicates per data point and R2 0 96 um 25 8007 50UM Chapter 5 Rev A 2003 G1 S G2 M cells N G2 M Sample S j of gate d 5 Control cells 23 6 S Mimosine treated cells 10 496 t E Colchicine treated cells 67 8 2 z 0 1000 575nm Area 5 6 2 Colchicine dose response Figure 5 15 shows a colchicine dose response curve for the G2M CCPM assay Image data were acquired 24 hours after addition of the inhibitor An ECs of 32 ng ml was calculated from the dose response curve Equivalent ECso values were obtained from analysis of the increase in mitotic cells and analysis of the disappearance of GO G1 S phase cells with increasing colchicine 50 75 a os 9 3 25 a GO G1 S F 50 e Mitosis os o Q D z o 25 0 10 10 Qs 10 10 Colchicine concentration ng ml e 14 Fig 5 15 Cells were imaged on the IN Cell Analyzer 3000 following a 2 hour treatment with 20 nM leptomycin B or untreated as a control Leptomycin B causes the reporter molecule to accumulate in the cell nuclei um 25 8007 50UM Chapter 5 Rev A 2003 5 6 3 Leptomycin inhibition of nuclear export L eptomycin B is an unsaturated branched chain fatty acid and is an important tool in the study of nuclear export It is a specific inhibitor of proteins containing a nuclear export signal 28 It has been reported that cyclin B1 translocation is inhibited by leptomycin B 29 The inh
37. cision in cancer Nat Rev Cancer 1 222 31 4 Walker M G 2001 Drug target discovery by gene expression analysis cell cycle genes Curr Cancer Drug Targets 1 73 83 5 Carnero A 2002 Targeting the cell cycle for cancer therapy Br J Cancer 87 129 337 6 Sampath D and Plunkett W 2001 Design of new anticancer therapies targeting cell cycle checkpoint pathways Curr Opin Oncol 13 484 90 7 Nurse P 2000 A Long Twentieth Century of the Cell Cycle and Beyond Cell 100 71 78 8 Thomas N and Goodyer I D 2003 Stealth sensors real time monitoring of the cell cycle Targets 2 1 pp26 33 9 Pines J 1999 Four dimensional control of the cell cycle Nature Cell Biology 1 E73 E79 10 Pines J and Hunter A 1989 Isolation of a human cyclin cDNA Evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2 Cell 8 833 846 11 Clute P and Pines J 1999 Temporal and spatial control of cyclin Bl destruction in metaphase Nature Cell Biology 1 82 87 12 Paiggio G Farina A Perrotti D Manni l Fuschi P Sacchi A and Gaetano C 1995 Structure and Growth Dependent regulation of the Human Cyclin Bl promoter Exp Cell Res 216 396 402 13 Hagting A Karlsson C Clute P Jackman M and Pines J 1998 MPF localization is controlled by nuclear export EMBO J 17 4127 4138 14 Hagting A Jackman M Simpson K and Pines J 1999 Tra
38. does not affect the normal cell cycle Fig 5 1 i CCPM cell line a U 2 OS wild type e 0 5 O Cc Q 1 0 re aL S 145 0 25 50 15 100 hours um 25 8010 50UM Chapter 5 Rev A 2003 9 2 Assay set up 9 2 1 General Assay Set up The cell cycle reporting cell line is an extremely flexible tool It is important to decide how to perform the assay to get maximum benefit from the product The assay can be performed as an endpoint or as a kinetic procedure and the cells can be imaged while still live or after fixation 5 2 1 1 End point method To facilitate analysis a nuclear marker is added This should be added at the end of the assay test period as many nuclear stains will prevent normal cell division and completion of M phase 9 2 1 2 Advantages of end point method e Relatively fast multiple images of the same cells are not required e Since each cell is only imaged once bleaching of the sample is not a concern This allows relatively high laser lamp powers and longer imaging times to be used This results in better image quality e Image analysis algorithms are available if using an IN Cell Analyzer 1000 or IN Cell Analyzer 3000 5 2 1 3 Disadvantages of end point method e When fine resolution of cell cycle position is required the relatively small proportion of prophase cells present necessitates imaging of over 1000 cells per condition to ensure statistically significant results e t can be difficul
39. ent expression have been identified 12 Human cyclin B1 mRNA appears at the end of the S phase and reaches its peak expression during G2 A 332 bp fragment upstream of the ATG codon Is negatively regulated in quiescent cells and its transcriptional activity depends on cell growth Specifically the region 150 58 is required for promoter inactivation during quiescence In the G2 M cell cycle marker technology the region from 150 to 180 is used to ensure S G2 specific expression of the reporter molecule 1 2 3 Cyclin B1 cytoplasmic retention sequence CRS Hagting et al constructed a cyclin B1 GFP fusion protein encoding construct that was microinjected either as pure protein or plasmid into HeLa and fibroblast cells 13 The expressed fusion protein behaved identically to the constitutive cyclin B1 that was detected with an antibody i e it was cytoplasmic for most of the cycle moved into the nucleus during prophase and was rapidly degraded toward the end of mitosis When the purified fusion protein was injected into the nucleus during S and G2 phases the protein was rapidly and completely exported from the nucleus within 10 minutes after injection The region containing the nuclear export signal was identified as an 11 amino acid hydrophobic stretch within the cytoplasmic retention sequence CRS 13 Nuclear export has been reported to be mediated by at least two different export factors exportin 1 CRM 1 which is required for t
40. eporting on epifluorescence microscopes For speed of screening and quality of the images obtained we recommend performing the cell cycle reporting assay on either the IN Cell Analyzer 1000 or the IN Cell Analyzer 3000 However it is possible to adapt the assay to be read and analyzed 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 3 NA 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 an environmental chamber are also recommended for assays performed on epifluorescence microscopes and a suitable software package will be required for image analysis Fig 5 10 shows the analysis from a kinetic assay where an individual G2MCCPM cell was imaged repeatedly over 60 hours and three mitotic divisions 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 maintenance Solent Scientific Portsmouth 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 An x y positioning stage with separate z focus Prior Scientific Cambridge
41. ficed in this manual Selection with flow cytometer Multiple selections of fluorescent and non fluorescent cell lines sequentially will tend to select cells with the desired characteristics Table 7 1 Quality control information for cell cycle position reporting cell line Fig 7 1 The intensity of GFP in the customer stocks of the stable cell line was compared to that in stable cells in culture at passages 9 and 24 There is no variation in the GFP intensity of the stable cells Table 7 2 Quality control information for the pCORON4004 CCEGFP expression vector um 25 8010 50UM Chapter 7 Rev A 2003 Chapter 7 Quality control 1 1 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 10 v v DMSO The cell line has the characteristics detailed in Table 7 1 Property Value Measurement method Viability from frozen 80 96 CASY1 Cell Counter and Analyzer System Model TT Cell diameter nm 15 18 CASY1 Cell Counter and Analyzer System Model TT Asynchronous cells fluorescence at gt 40 000 for 20 3 x 10 cells per ml passages after RFU dispatch FARCyte Gain 61 Flow Cytometry analysis The supplied cell line was analyzed by flow cytometry to look at the variation in the intensity of GFP in the stable cell population compared to untransfected control cell population Figure 7 1
42. he export of the HIV Rev protein and IxBo and exportin2 CAS which is responsible for the export of importin A Nuclear export via exportin 1 is inhibited by the drug leptomycin B 13 and evidence implicates this pathway in the export of cyclin B1 When cells are treated with leptomycin B cyclin B1 accumulates in the nucleus showing that there is constitutive nuclear import occurring The CRS of Xenopus cyclin B1 has been shown to bind to exportin 1 14 Phosphorylation of the CRS is required for nuclear trafficking of cyclin Bl 14 During S and G2 phase cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export At M phase cyclin B1 is phosphorylated in the CRS The nuclear export sequence a region of the CRS is then inactivated and the protein moves rapidly to the nucleus Figure 1 3 The import of cyclin B1 into the nucleus occurs approximately 10 minutes before breakdown of the nuclear envelope as the cells enter prophase the first stage of mitosis 1 2 4 Cyclin B1degradation box D box Cyclin dependent kinases CDKs promote progression through the cell cycle By synthesizing and degrading CDK activators and inhibitors the cell can be made to progress through the cell cycle and directly trigger the transition from metaphase to anaphase King et a 15 describe how two distinct ubiquitin conjugation pathways mediate proteolysis during the cell cycle One pathw
43. ibition is thought to involve direct binding to CRMI which prevents binding of CRMI to proteins containing the nuclear export signal 30 31 Figure 5 16 shows the effects of 20 nM leptomycin B on the cell cycle position reporter cell line The compound inhibits the export of the reporter molecule confirming that the reporter molecule is continuously shuttling between the nucleus and cytoplasm Control Leptomycin treated ee Chapter 6 Vector use details The plasmid vector pCORON4004 CCEGFP Fig 3 1 can be used to transiently or stably express the cell cycle reporting fusion protein in the cell line of choice 6 1 General guidelines for vector use pCORON4004 CCEGFP has been used successfully to express the cell cycle reporting fusion protein transiently in MCF7 HeLa A431 and U 2 OS cell lines and stably in the U 2 OS derived cell line Expression levels and other assay parameters may vary depending on the chosen parent cell line and transfection procedure 6 2 Transient transfection with pCORON4004 CCEGFP Transfection protocols must be optimised for the cell type of choice Both choice of transfection reagent and cell type will affect efficiency of transfection FUGENE 6 Transfection Reagent Roche has produced successful results with pbCORON4004 CCEGFP for a variety of cell lines The following standard in house protocols for adherent cells may serve as useful guidelines for establishing an appropriate protocol For more informa
44. included with this assay The vials are stored frozen in vapor phase liquid Nitrogen um 25 8010 50UM Chapter 5 Rev A 2003 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 min Do not thaw the cells for longer than 3 min as this decreases viability 3 Remove the cryo vial from the water bath and wipe it with 70 v v Ethanol Immediately transfer the cells under asceptic conditions to a T flask containing 10 20 ml Growth medium depending on size of T flask at 37 C NOTE To ensure maximum cell viability do not allow the cells to thaw at room temperature and do not thaw the cells using your hand to warm the vial 5 1 4 Cell sub culturing procedure Incubation 5 CO5 95 humidity 37 C Passage ratio 1 5 to 1 20 twice a week The cells should be passaged when they reach 70 to 95 confluence All reagents should be warmed to 37 C 1 Aspirate the medium from the cells and discard 2 Wash the cells with 10 20 ml PBS depending on flask size 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 trypsin EDTA 2 ml for T 75 flasks and 5 ml for T 175 flask ensuring that all cells are in contact with the solution Remove the trypsin solution Wait for 3 10 min for the cells to round up loosen Check on an inverted microscope 5 When the cells are
45. ing the IN Cell Analyzer 3000 system and software Mimosine treatment increases the percentage of cells in GO Gl and S phases and decreases the number of cells in G2 M Colchicine treatment increases the percentage of cells in G2 M but decreases the percentage of cells in GO G1 and S phases To allow comparison of IN Cell Analyzer 3000 results with flow cytometry data the percentage of cells in G2 M was defined as the sum of the percentages of cells classified as G2 prophase or mitosis by the Cell Cycle Trafficking Analysis Module Data bars represent the mean values obtained from 32 wells with error bars showing standard deviation um 25 8007 50UM Chapter 5 Rev A 2003 5 6 Assay characterization 5 6 1 Cell cycle validation As part of the assay and image analysis validation process G2M CCPM assay results obtained using the IN Cell Analyzer 3000 system and software were compared with results obtained by flow cytometry analysis of the G2M CCPM cell line Sample preparations for each experiment were prepared in parallel from the same batch of G2M CCPM cells For analysis using the IN Cell Analyzer 3000 cells were incubated in the absence or presence of cell cycle inhibiting compounds colchicine or mimosine as described in section 5 5 Following the incubation period cells were imaged live and analyzed using the Cell Cycle Trafficking Analysis Module for flow cytometry analysis cells were treated fixed and stained as
46. ins to activate CDC2 and drive the cells into mitosis these mutations dominantly arrest cell division in telophase If the cyclin B D box is grafted onto otherwise stable proteins those proteins become unstable in mitosis i e the D box is portable We have exploited this property of the cyclin Bl D box to effect degradation of EGFP specifically at the end of metaphase A P CDC2 ES P Cyclin B1 X JOC Cyclin B1 P A CDC Pipe Cyclin E A e P r3 LH L wore 0 Cyclin B1 p Cdc25 p P CDC2 Cyclin B1 Cyclin B1 ech OC e e a CDC2 1 3 Applications in drug discovery Since the CCPM reporter does not interfere with the cell cycle of the host cell it allows non destructive measurement of cell cycle position The CCPM sensor has many potential applications in cultured cells and in more complex model systems Perhaps most significantly CCPM expression in stable cell lines allows continuous and individual monitoring of the cell cycle status of every cell in the culture and consequently removes the need to work with synchronized cells to identify cell cycle related phenomena One use of a stable cell line expressing the CCPM construct is to screen for anti mitotic or anti proliferative compounds that would be expected to arrest a proportion of cells in a certain phase of the cell cycle Another potential Fig 1 4 Cell cycle dependent CCPM reporter expression and location There are four different patterns that can
47. ipt 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 recover the contents 4 3 2 Cells Care should be taken to ensure that the cells are not warmed if they are not being used immediately Do not centrifuge the cell samples upon thawing um 25 8010 50UM Chapter 5 Rev A 2003 Chapter 5 Cell assay design 5 1 Culture and maintenance of U 2 OS derived cell cycle reporting cell line 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 M 8403 e Fetal Bovine Serum FBS Invitrogen life technologies 10099 141 or equivalent Heat inactivated e Penicillin Streptomycin P S 5000 units ml penicillin G sodium and 5000 ug ml streptomycin sulfate Invitrogen life technologies 15070 063 or equivalent e Geneticin 6418 Sigma G 7034 or equivalent e Irypsin EDTA 1x in HBSS w o calcium or magnesium Invitrogen life technologies 25300 054 or equivalent e PBS Dulbecco s w o calcium magnesium or sodium bicarbonate Invitrogen life technologies 14190 094 or equivalent e
48. is a trademark of Microsoft Corporation FACS is a trademark of Becton Dickinson and Co Oracle is a trademark of Oracle Corporation Hoechst is a trademark of Aventis Geneticin is a registered trademark of Life Technologies Inc DRAQ5 is a trademark of Biostatus Limited Amersham Biosciences UK Limited 2003 All rights reserved http www amersham com Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Fig 3 1 Vector map of pCCORON4004 CCEGFP expression vector um 25 8010 50UM Chapter 3 Rev A 2003 Chapter 3 Product contents 3 1 Components summary e pCORON4004 CCEGFP expression vector 1 vial containing 10 ug DNA Supplied in TE buffer 10 mM Tris 1 mM EDTA pH8 O NIF2034 e Cell cycle reporting cell line U 2 OS derived 2 vials each containing 1 x 106 cells in 1 ml of fetal calf serum NIF2033 e User manual 3 2 GFP expression vector pCORON4004 CCEGFP NIF2034 The supplied plasmid pCORON4004 CCEGFP is 6 0 kb in length and contains a bacterial ampicillin resistance gene and a mammalian neomycin resistance gene The cell cycle specific cyclin B1 promoter fragment can be excised from the vector using EcoRI and Hindlll The cyclin B1 N terminus EGFP fusion
49. loose dislodge the cells by adding Growth medium 5 ml for I 75 and 10 ml for T 175 and resuspend by gentle agitation with a 10 ml pipette until all clumps have dispersed 6 Aspirate the cell suspension and dispense the cells into a new culture vessel containing the amount of Growth medium required to obtain the desired passage ratio 5 1 5 Cell seeding procedure This procedure is for cells grown in a standard T 175 flask and seeded into microplates All reagents used for seeding cells should be pre warmed to 37 C l Aspirate the medium from the cells and discard Wash once with PBS 2 Add 5 ml trypsin EDTA to the cell surface and leave at 37 C for approximately 3 10 min or until the cells loosen easily Tap the flask gently to dislodge the cells 3 Add 5 ml Growth medium and gently resuspend the cells using a 10 ml pipette 4 Spin the cell suspension at 1000rpm for 5 minutes and decant off the supernatant 5 Resuspend cell pellet in 5 10 ml growth media 6 Count the cells using either a CASY1 Cell Counter and Analyzer System Model TT or a hemocytometer Fig 5 1 Growth curve of U 2 OS derived cell lines Untransfected cells and cells expressing the cell cycle position marker have indistinguishable growth characteristics Both cell lines have a doubling time of 24 hours R 0 999 um 25 8010 50UM Chapter 5 Rev A 2003 7 Using fresh Growth medium adjust the cell density so that it will deliver the desi
50. lute the test compounds and dispense onto the cells Each well should contain a final volume of 100 ul 3 Incubate the microplates at 37 C 5 CO for 24 hours In this time the majority of cells would be expected to complete a cell cycle In the presence of a cell cycle perturbing drug the normal ratio of cells in each phase will be altered 4 Remove contents of wells by inverting plate onto a pad of sterile tissues and blot gently 5 Add 200 ul well of 4 formalin and incubate at room temperature in the dark for 30 minutes 6 Remove contents of wells and wash plate once with 100 ul PBS 7 Dilute the nuclear stain DRAQ5 or Hoechst to 1 uM and add 100 ul well Incubate at 37 C for 30 minutes 8 Remove contents of wells and wash plate twice with 100 ul PBS 9 Leave the cells in 100 ul of PBS and seal the plate to prevent evaporation Store the plate at 4 8 C in the dark until ready to image Plates stored in this manner are stable for at least 2 weeks 5 2 5 Kinetic assay for cell cycle perturbing drugs 1 Seed 3000 cells per well in 100 ul of Growth medium and incubate for 24 hours at 37 C 5 CO 2 Remove media from the plates by inversion and blotting onto a pad of Sterile tissues Prepare control wells containing either Growth medium 2 mM mimosine see section 5 1 2 or O 1 ug ml colchicine in Growth medium Dilute the test compounds and dispense onto the cells Each well should contain a final volume of 100 ul
51. nd the ratio of the two values are determined um 25 8010 50UM Chapter 5 Rev A 2003 9 3 1 2 End point assay using the IN Cell Analyzer 3000 Images obtained from the IN Cell Analyzer 3000 are shown in Figure 5 4 The control image shows a few cells in mitosis The number of mitotic cells Is increased by treatment with colchicine and reduced by treatment with mimosine Control Colchicine cells treated cells Mimosine treated cells 9 3 1 3 End point assay analysis using the IN Cell Analyzer 3000 On the IN Cell Analyzer 3000 the location and intensity of the cell cycle phase marker fusion protein can be determined using the Cell Cycle Trafficking analysis module The number of cells in each of four cell cycle stages Is then reported A detailed description of this algorithm is provided in the Cell Cycle Trafficking Analysis Module user manual The following is a brief description of the algorithm and its output The algorithm identifies the intensity of a region of the cytoplasm lcyt the intensity of the nucleus Inuc and the ratio between the nuclear and cytoplasmic intensities nuc cyt Figure 5 5 Average pixel intensity in nuclear region gives Inuc Average pixel intensity in the ring around the nuclear gives Icyt Ratio of nuclear to cytoplasmic intensities gives nuc cyt Fig 5 6 Four different phases of the cell cycle are distinguished by examining Inuc and l1 ratiol Fig 5 7 Data from
52. nslocation of cyclin B1 to the nucleus at prophase requires a phosphorylation dependent nuclear import signal Current Biology 9 680 689 15 King R W Deshaies R J Peters J M and Kirschner M W 1996 How Proteolysis Drives the Cell Cycle Science 274 1652 1659 16 Okazawa M et al 1998 Endothelin induced apoptosis of A375 human melanoma cells J Biol Chem 273 20 12584 92 17 Abel A et al 2000 Cell cycle dependent coupling of the vasopressin Vla receptor to different G proteins J Biol Chem 275 42 32543 51 18 Bestervelt L et a 2000 Divergent proliferative responses to a gastrin receptor ligand in synchronized and unsynchronized rat pancreatic ARA2J tumour cells Cell Signal 12 1 53 61 19 Chernikova S B et al 2001 Cell cycle dependent effects of wortmannin on radiation survival and mutation Radiat Res 155 6 826 31 20 Gorodetsky R et a 1998 Paclitaxel induced modification of the effects of radiation and alterations in the cell cycle in normal and tumor mammalian cells Radiat Res 150 3 283 91 21 Ponten J et al Two established in vitro cell lines from human mesenchymal tumours Int J Cancer 2 434 447 1967 22 Heldin C H et al A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A chains Nature 319 511 514 1986 23 Raile K et al Human osteosarcoma U 2 OS cells express both insulin like growth factor IGF I
53. r contact with skin or clothing 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 um 25 8010 50UM Chapter 4 Rev A 2003 with a suitable disinfectant 5 Hands should be washed after using genetically modified 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 4 2 Storage The pCORON 4004 CCEGFP expression vector NIF2034 should be stored at 15 C to 30 C The U 2 OS derived cells expressing the GFP fusion protein NIF2033 should be stored at 196 C in liquid Nitrogen vapour 4 3 Handling Upon rece
54. red number of cells to each well For example to plate 3000 cells per well in 100 ul of suspension the suspension is adjusted to 3 x 104 cells per ml 8 Incubate the plated cells for 24 h at 37 C 5 CO before starting the assay 9 If the cells are near confluence prior to trypsinization they should be passaged into two T flasks They will then be ready for seeding the following day 5 1 6 Cell cryopreservation procedure 1 Harvest the cells as described in section 5 1 4 and prepare a cell suspension containing 1 x 109 cells per ml 2 Pellet the cells at approximately 1000 g for 5 min 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 109 cells in 1 ml of Freeze medium 4 Transfer the vials to a cryo freezing device and freeze at 80 C for 16 24 h 5 Transfer the vials to the vapor phase in a liquid nitrogen storage device 9 1 7 Growth characteristics Under standard growth conditions the cells should maintain an average size of 16 5 um as measured using a CASY1 Cell Counter and Analyzer System Model TT The doubling time of the stably expressing cell line in exponential growth phase has been determined to be approximately 24h under standard conditions This is identical to wild type U 2 OS cells grown at the same time and under the same conditions confirming that expression of the reporter molecule
55. required 4 2 Seed cells at a lower density 4 3 Mitotic and rounded cells have a smaller surface area in contact with the Shading across image field IN Cell Analyzer 3000 only Q Low cell number when using mitotic inhibitors e g colchicine um 25 8010 50UM Chapter 8 Rev A 2003 plate Poly L Lysine coat the plate to increase adherence Possible cause 5 1 Flat field correction not applied or flat field solution too weak Remedy 5 1 Apply flat field correction or adjust flat field solution Possible cause 6 1 Cells lost when mixing contents of well with a pipette after addition of nuclear marker e g after DRAQS addition 6 2 Decanted contents of plate after treatment with a mitotic inhibitor e g colchicine Remedy 6 1 Do not mix cells treated with a mitotic inhibitor e g colchicine with a pipette after nuclear dye e g DRAQS addition Mix these wells by tapping or swirling the plate gently 6 2 After cells have been treated with a mitotic inhibitor e g colchicine do not remove contents of well prior to reading the assay um 25 8010 50UM Chapter 9 Rev A 2003 Chapter 9 References 9 1 References 1 Nurse P 2000 The Incredible Life and Times of Biological Cells Science 289 1711 1716 2 Evan G and Littlewood T 1998 A matter of life and cell death Science 281 1317 22 3 Malumbres M and Barbacid M 2001 To cycle or not to cycle a critical de
56. s Location of the reporter is controlled by the cytoplasmic retention sequence CRS from Cyclin B1 which localizes the reporter to the cytoplasm until the start of mitosis when it translocates to the nucleus um 25 8010 50UM Chapter 1 Rev A 2003 1 2 Cell Cycle Phase Markers The G2M cell cycle phase marker G2MCCPM assay employs a non destructive dynamic GFP based probe to report the position of individual cells in the cell cycle 8 The probe Figure 1 2 is comprised of cell cycle dependent expression destruction and localization elements from the gene for cyclin B1 a tightly regulated cell cycle dependent kinase that is expressed in late S phase and is subsequently degraded during mitosis Figure 1 3 By quantifying the location and fluorescence intensity of the expressed reporter molecule the cell cycle position of individual cells can be pinpointed to one of four distinct phases of the cell cycle Figure 1 4 1 2 1 Cyclin B1 function Cyclins are a family of proteins that bind to and activate cyclin dependent kinases Cyclins are produced at specific times during the cell cycle and their expression levels and location are tightly controlled 9 Cyclin B1 was the first human cyclin identified and is a 62 kDa protein encoded by a 1 6 kb mRNA 10 It is synthesized during the late S and G2 phases and complexes with the cell cycle dependent kinase p34cdc2 The key point in the transition of a cell from G2 to mitosis i
57. s the activation of a protein serine threonine kinase which has been variously identified as MPF in frog eggs as histone H1 kinase in sea urchin eggs and as growth associated histone H1 kinase in mammalian cells All these activities have a common component a 32 34 kD protein which in the yeast S pombe is the product of the cdc2 gene and in S cerevisiae is encoded by CDC28 The protein is sufficiently well conserved that antibodies raised against conserved epitopes cross react with all the species tested The human homolog of this protein p34cdc2 was identified by its ability to complement cdc2 in yeast The cdc2 kinase activity has been shown to vary through the cell cycle even though the level of the protein itself does not change In HeLa cells cdc2 kinase activity is absent in Gl and increases through S G2 and M phases in a manner that correlates with its association to cyclin B1 11 The p34cdc2 binding site is located at the C terminus of cyclin B1 We have deliberately excluded this region from our fusion protein so that the reporter molecule will not bind or activate p34cdc2 and therefore will act as a stealth sensor that does not affect the cell cycle cyclin Bl cyclin Bl promoter N terminus EGFP S M H _ E Be D Box CRS um 25 8010 50UM Chapter 1 Rev A 2003 1 2 2 Cyclin B1 promoter The human cyclin B1 promoter has been well characterized and the regions that are essential for cell cycle depend
58. s the complementary strand 2957 4045 4087 c 4240 c 5016 926 2606 c 2678 c 2742 c 3257 3789 149 c 1733 1826 c 783 926 1055 1304 1495 2157 3052 3145 3219 3409 3471 3917 4414 4499 5660 1918 3887 4137 4242 5250 2993 c 3374 3824 32 155 247 315 449 469 977 1037 1050 1094 1103 1957 1987 2019 2021 2063 2090 2120 2657 2729 2780 2859 2865 3097 3181 3204 3343 3349 3466 3502 3549 3816 3912 4964 5352 5924 5972 2084 c 3719 c 3773 4240 c 3339 4848 5022 c 161 1036 1603 2123 3425 3606 4997 1595 591 646 c 652 c 664 c 1497 c 2317 2797 c 3050 3251 4437 4607 c 4876 4919 5037 5443 5555 c 5568 c 3503 3 88 74 79 141 355 393 931 1056 1168 1243 1297 2626 2681 2698 3493 5811 5824 5945 26 130 132 452 1214 1532 2000 2024 2044 2420 2507 3172 3473 3505 3906 3986 4089 4091 4191 4523 5016 5346 5927 13827 889 1385 2509 3277 3523 3900 4435 4452 5220 5232 5318 5329 5982 343 45 51 132 381 445 452 1175 1216 1532 1978 2002 2015 2024 2046 2072 2080 2917 3100 3108 3172 3209 3475 3505 3507 3735 3988 4091 4191 4523 4860 4953 5346 5455 5629 5729 5796 161 1036 1603 2123 3425 3606 4997 1918 3887 3736 631 1314 1596 1630 2563 3411 3924 4600 236 343 370 490 659 683 695 725 1489 1507 2570 2872 3769 3920 4155 4581 5121 52367 5696 135 430 863 891 1387 1535 1573 1917 1921 1957 2511 3279 3357 3438 3447 3525 3902 4401 4437 4454 4712 4758 4776 5117 5222 5234 5312 5320 5331 5406 5984 um 25 8010 50UM Chapter 11 Rev A 2003 Enzyme
59. s were not stored properly or they are out of date 1 6 Cells have been stressed during assay Remedy 1 1 Start a 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 kit 1 2 Verify density of cell plating adjust plating density to values that yield optimal assay response 1 3 Check that the primary parameters are correct and suitable for the cells currently in use 1 4 Ensure that proper incubation is maintained as consistently as possible during the assay When plates are out of the CO incubator for extended periods it is essential that HEPES buffer is added to the medium to maintain proper pH 1 5 Repeat assay with fresh reagents 1 6 Use actively growing cells maintained at 37 C Pre warm reagents to o Possible cause 2 1 Nuclear stain concentration too low 2 2 Nuclear stain incubation time too short Remedy 2 1 Adjust Nuclear stain concentration to recommended level 2 2 Adjust Nuclear stain incubation time to recommended length Possible cause 3 1 Autofocus Offset is chosen incorrectly or the system may need to be realigned Remedy 3 1 Alignment and calibration of instrument Perform Z stack on cells Change Autofocus Offset Possible cause 4 1 Plate is not treated correctly 4 2 Plating density too high 4 3 Cell cycle drugs block in mitosis Remedy 4 1 Poly L Lysine coat the plate if
60. t 1104769 0804457 and Japanese patent JP3283523 and other pending and foreign patent applications Columbia University under US patent Nos 5 491 084 and 6 146 826 Rights to use this product as configured are limited to internal use for screening development and discovery of therapeutic products NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rights are conveyed University of Florida Research Foundation under US patents 5 968 750 5 8 4 304 5 795 737 6 020 192 and other pending and foreign patent applications Cancer Research Campaign Technology Limited under patent publication number WO 03 031612 and other pending and foreign patent applications The exact terms of use for the product as configured are specified in the license accompanying the product but are limited to internal use for screening development and discovery of therapeutic products No rights other than those expressly granted are conveyed All goods and services are sold subject to terms and conditions of sale of the company within the Amersham Biosciences group which supplies them Copies of these terms and conditions are available on request Amersham and Amersham Biosciences are trademarks of Amersham plc Biolmage is a trademark of Biolmage A S Imaging Research is a trademark of Imaging Research Inc Biocarta is a trademark of Biocarta Inc um 25 8010 50UM Chapter 2 Rev A 2003 FuGENE is a trademark of Fugent LLC Microsoft
61. t to differentiate cells undergoing mitosis and necrosis when only one image at a single time point is captured Drugs that arrest cells in mitosis can be mistaken for toxic compounds and vice versa leading to the potential for false positive or negative results 5 2 1 4 Kinetic method No nuclear marker is used and the only signal measured is from EGFP itself The absence of nuclear marker allows the cells to divide repreatedly 9 2 1 5 Advantages of kinetic method e As the progress of cells is followed over time it is possible to obtain a large amount of information from a relatively small number of cells For example it is possible to determine whether a rounded cell is in mitosis because it should become dim and split into two daughter cells or whether it is dying It is also possible to observe cells in prophase by following a relatively small number of cells but imaging frequently e Processes that can not be accurately spotted in an end point assay e g unusual drug induced appearance or disappearance of the reporter molecule are observable 5 2 1 6 Disadvantages of kinetic method e Automatic image analysis algorithms are not currently available e Photo bleaching of the reporter or phototoxicity can result as a consequence of repeated exposure of the cells to high energy light sources The consequent use of neutral density ND filters may reduce the Fig 5 2 A typical plate map for a cell cycle inhibitor screen Control
62. tion refer to manufacturer s guidelines for the desired transfection reagent 6 2 1 FUGENE 6 Transfection Reagent protocol Day 1 Seed cells so that the density will be 50 80 the next day Day 2 e Add serum free McCOYS 5A media to an empty tube e Add FuGENE 6 Transfection Reagent directly into this medium dropwise Mix by gentle pipetting e Add the FuGENE 6 Transfection Reagent medium mix to the tube containing the DNA Mix by gentle pipetting e Incubate for a minimum of 15 min at room temperature e Add transfection mixture directly to the cells dropwise without changing the medium and mix by swirling gently Day 3 Change media to a complete McCOYS 5A 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 Sigma G 7034 um 25 8010 50UM e Chapter 6 Rev A 2003 um 25 8010 50UM Chapter 6 Rev A 2003 6 3 Stable cell line generation with pCORON4004 CCEGFP The process of establishing stable cell lines involves a large number of variables many of which are cell line dependent Standard methods and guidelines for the generation of stable cell lines are widely available in the public domain 32 pCORON4004 CCEGFP has been used to generate stably transfected cell populations The magnitude of the response with different cell lines are unknown and may deviate considerably from the values speci

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