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EGFP-2x FYVE Assay - GE Healthcare Life Sciences

1. Control or Methanol on the EGFP 2x FYVE ME Wortmannin translocation Error SD n 8 008 replicates per data point x T T g aso a S 2 2005 5 O 150 5 E 0 ia a oa fal ain 0 0 1 0 25 0 5 1 0 2 0 DMSO Control EE Wortmannin 300 5 x x T x 2504 I me _ 2004 wn 5 5 150 5 8 507 o 5 g 0 ro fat 4 5a ma H 0 0 1 0 25 0 5 1 0 2 0 EtOH Control EE Wortmannin 300 5 2504 T me a S 2004 cw SE 150 5 2 507 o OD ou wn g 0 pame Ea a n Pa 0 0 1 0 25 0 5 1 0 2 0 MeOH 25 8010 21UM Chapter 5 Rev B 2006 20 5 4 7 Effect of different assay media To determine the effect of varying the assay media on Wortmannin induced EGFP 2x FYVE fusion protein translocation reporter cells were assayed in either Nutrient Mixture F 12 Ham or KrebsRingerWollheim media with a range of additives 10 mM HEPES BSA and FBS KrebsRingerWollheim media consists of 140 mM NaCl 3 6 mM KCI 0 5 mM NaH PO H 0 0 5 mM MgSO 7H 0 2 mM NaHCO 1 5 mM CaCl 2H O 6 mM D Glucose and 10 mM HEPES The results shown in Fig 5 9 demonstrate that the assay tolerates a range of assay media Sin MEE Control Fig 5 9 The effect of different assay I HB Wortmannin media
2. Cell density per well 5 4 4 Wortmannin dose response Fig 5 6 shows a dose response curve for the supplied cells to Wortmannin The data were collected 30 minutes after addition of Wortmannin and demonstrate an ECeg of 1 88 nM 300 Fig 5 6 Wortmannin dose response curve using the supplied EGFP 2x 200 J FYVE cell line The calculated EC was 1 88 nM Error SD n 8 replicates per data point 100 4 Translocation Index Fgrains 0 T T T 1 on 10 10 109 108 107 106 Wortmannin M 25 8010 21UM Chapter 5 Rev B 2006 19 5 4 5 Time course Fig 5 7 shows a typical time course of the translocation and indicates that the maximal translocation occurs after 30 minutes of treatment with 100 nM Wortmannin Fig 5 7 Time course of EGFP 2x x Wortmanin FYVE translocation in response to Wortmannin treatment Maximal response is seen after 30 minutes Error SD n 24 replicates per data point Wortmannin n 16 replicates per data point control Translocation Index Fgrains 0 T T T T T 1 0 25 50 75 100 125 150 Time minutes 5 4 6 Sensitivity of assay to DMSO Ethanol and Methanol The EGFP 2x FYVE translocation was measured in the presence of DMSO lt 2 Ethanol lt 2 or Methanol lt 2 As can be seen in Fig 5 8 the assay is stable to the presence of upto 0 5 DMSO Ethanol or Methanol Fig 5 8 Effect of DMSO Ethanol
3. Sall 2341 Sap 2 4133 c 4343 c Sau3Al Sa 662 747 1607 1755 1793 1834 2101 2598 2602 2638 3192 3960 4038 4119 4128 4206 4583 5082 5118 5135 5393 5439 5457 5798 5903 5915 5993 6001 6012 6087 6665 Sau96l 13 237 430 1280 1681 1764 2629 2917 4305 4785 5401 5623 5640 5719 Scal 4 1064 2011 2257 5284 ScrFl 28 244 437 1137 1153 1278 1390 1465 1519 1791 1969 2215 2347 2348 2607 3309 3364 3381 3793 3953 4176 4693 4728 5229 5580 6276 6494 6507 6628 SexAl 1 3362 SfaNl 26 511 c 1206 c 1484 1499 1598 1857 c 2042 2103 c 2288 2438 c 3130 c 3170 3352 3424 3747 c 4002 c 4088 4152 4218 c 4427 4611 c 4705 5064 c 5313 5504 c 6556 c Sfcl 9 835 1080 1926 2172 2688 3838 5519 6197 6388 Sfil 1 3548 Sful 1 4471 Smal 1 2348 SnaBl 1 494 Snol 3 4596 5093 6339 Spel 1 153 Sphl 3 3343 3415 4194 SspBl 2 97 1817 Sspl 4 6 53 3119 4960 Stul 1 3594 Styl 7 514 1106 1856 3206 3502 3595 4221 25 8010 21UM Chapter 11 Rev B 2006 32 Enzyme ofcuts Positions c indicates the complementary strand Taq 25 824 945 1157 1451 1478 1493 1622 1839 2036 2282 2342 2360 2601 2876 3638 3902 4058 4082 4118 4280 4471 4570 5111 6555 6660 Tfil 4 3617 4274 4408 4567 Thal 23 214 1436 1754 1851 2051 2297 2683 2707 2727 3103 3190 3855 4156 4188 4589 4669 4772 4774 4874 5206 5699 6029 6610 Tru9 27 161 784 830 849 917 1052 1067 2494 2555 2701 2972 3070 3087 3098 3110 3121 3644 4633 4814 5186 5551 5590 5825 5878 5892 5897 5949 Tsp509 15 172 786 1040 1843
4. The cells should be seeded in the appropriate microplate the day before the experiment The Growth medium is decanted the cells washed and Assay medium added to each well Test compound and controls are added to required wells After 30 minutes incubation the microplates are placed into the IN Cell Analyzer 3000 The Granularity Analysis Module is used to analyze each well Seed cells Incubate overnight 37 C 5 CO Decant Wash Decant Add Assay medium with nuclear stain Add test compounds and controls Incubate 30 minutes 37 C 5 CO gt Image plates on the IN Cell Analyzer 3000 Analyze using Granularity Analysis Module Remove from IN Cell Analyzer 3000 STOP 5 2 4 Assay protocol 96 well format NOTE whenever possible keep the microplate at 37 C 5 CO and 95 humidity 1 The day before starting the assay seed 0 6 x 104 cells per well in 200 ul of Growth medium Incubate for 24 hours at 37 C If one of the wells on the cell plate is used for flat field correction it should not contain cells 2 On the day of the assay prepare the test compounds solvent controls if used and reference control e g Wortmannin These samples are typically prepared at four fold of the final concentration in Assay medium For Wortmannin a final 25 8010 21UM Chapter 5 Rev B 2006 16 Fig 5 2 Flow diagram showing a basic protocol suitable for a screen to identify PI3 Kinase inhibitors All incuba
5. 1 2 Using this assay format Wortmannin has a typical EC value of 1 9 nM Fig 1 2 Wortmannin induced redistribution of EGFP 2X FYVE from the early endosomal membranes to the cytoplasm PI3K inhibitor 30 minutes pP Un treated cell Treated cell EGFP 2x FYVE is EGFP 2x FYVE is concentrated in redistributed to the endosomes cytoplasm 25 8010 21UM Chapter 1 Rev B 2006 5 2 Licensing considerations 2 1 Right to use Use of this assay is limited as stated in the terms and conditions of sale These vary in accordance with the product code purchased Description Product Code EGFP 2x FYVE Assay Screening applications 25 8010 21 EGFP 2x FYVE Assay Research applications 25 8010 22 EGFP 2x FYVE Assay 6 month assay evaluation 25 8010 23 EGFP 2x FYVE Assay 12 month assay evaluation 25 8010 24 This assay was developed in collaboration with Biolmage A S and is sold under license from Biolmage A S under patents US 6172188 US 5958713 US6518021 EP 851874 EP 0815257 EP 0986753 and other pending and foreign patent applications and Invitrogen IP Holdings Inc formerly Aurora Biosciences Corporation under US patents US 5625048 5777079 5804387 5968738 5994077 6054321 6066476 6077707 6090919 6124128 6319969 6403374 European Patent 1104769 0804457 and Japanese patent JP 3283523 and other pending and foreign patent applications and Columbia University This product is also sold under license from Co
6. 3258 c 3443 3455 3464 3476 3486 3497 3543 3698 3761 3855 c 3919 c 4020 c 4023 c 4263 4303 c 4308 4358 c 4374 4400 4456 c 4515 4587 4625 4651 4661 4700 4874 c 4921 5020 c 5129 c 5206 c 5250 5371 c 5417 5608 c 5699 c 6061 6070 c 6205 6315 c 6436 c 6455 c 6582 c 6610 c Acsl 4 1843 2440 3094 3105 Acyl 8 276 329 412 598 3789 4491 4843 5225 Aflll 4 829 848 1051 3643 Afilll 1 1849 Agel 1 1095 Alul d 728 759 834 1048 1128 1161 1233 1266 1482 1530 1641 1815 2076 2322 2479 2824 3081 3271 3559 3613 3895 4353 4714 4733 5412 5475 5575 6096 6353 6399 6489 Alw44l 3 4596 5093 6339 Alwl 20 1602 c 1801 2096 c 2109 2597 c 2606 3200 3968 4033 c 4214 4578 c 4591 5126 5130 c 5447 5910 c 5911 6007 c 6009 6095 AlwNI 1 6244 Aosl 4 2660 3199 3891 5542 ApaLl 3 4596 5093 6339 Apol 4 1843 2440 3094 3105 Asel 2 161 5590 Asn 2 161 5590 Asp700 3 1998 2244 5165 Asp718 1 3245 AspEl 1 5765 AspHI 8 730 1717 3902 4092 4600 5097 5182 6343 Aspl 1 3907 Asull 1 4471 Aval 2 1838 2346 Avall 4 1764 4305 5401 5623 Avill 4 2660 3199 3891 5542 Avril 1 3595 BamHI 2 2101 4583 Banl 8 619 977 1143 2870 3245 3788 3823 5812 Banll 7 730 1893 2063 2139 2309 2840 4154 Bbs l 1 962 25 8010 21UM Chapter 11 Rev B 2006 28 Enzyme ofcuts Positions c indicates the complementary strand Bbvl 30 821 c 1253 c 1359 1643 1650 1676 c 1679 c 1866 c 1912 c 2085 2112 c 2158 c 2331 2466c 2673 2741 3212 3736 c 3862 3904 3920c 40
7. 4092 4843 5163 5536 5952 Maelll 21 215 302 651 839 902 1290 1779 2465 2721 2733 3909 4215 4716 5104 5292 5445 5503 5834 6117 6233 6296 Maml 2 2597 4582 Mbol 1 662 747 1607 1755 1793 1834 2101 2598 2602 2638 3192 3960 4038 4119 4128 4206 4583 5082 5118 5135 5393 5439 5457 5798 5903 5915 5993 6001 6012 6087 6665 Mpboll 20 967 1350 c 1395 c 1398 c 1593 1994 c 2240 c 2636 2772 c 3612 c 4150 4360 4440 c 4983 5092 5170 5925 5996 c 6148 c 6660 c Mcrl 8 665 1100 2355 2641 3698 5247 5396 6319 Mfel 1 2504 Mlul 1 1849 MIuNI 3 11 65 3871 Mnill 41 703 c 870 c 1116 c 1197 c 1203 c 1297 1434 c 1446 c 1497 c 1617 c 1905 c 2037 c 2151 c 2283 c 2540 c 2580 2620 c 2884 3224 c 3232 3248 c 3526 c 3532 c 3556 3562 3569 c 3572 c 3584 c 3704 c 3840 c 4197 c 4390 4739 c 4798 5392 c 5598 c 5745 5826 6226 6476 c 6550 Mrol 1825 Mscl 5 11 65 3871 Msel 27 161 784 830 849 917 1052 1067 2494 2555 2701 2972 3070 3087 3098 3110 3121 3644 4633 4814 5186 5551 5590 5825 5878 5892 5897 5949 Msll 12 519 1108 1138 1288 1465 1594 4226 4508 4547 4994 5353 5512 MspA1l 10 1925 2076 2171 2322 3271 3895 4663 5129 6070 6315 Mspl 30 1096 1136 1199 1259 1790 1826 1968 2214 2347 2606 2807 3694 3771 3793 3821 3952 4042 4109 4290 4693 4727 5228 5470 5580 5647 5681 6085 6275 6301 6448 Munl 1 2504 Mval 14 244 437 1153 1278 1390 1465 1519 3309 3364 3381 4176 6494 6507 6628 Mvnl 23 214 1436 1754 1851 2051 2297 2683 2707 2727 3103 3190 3855 4156
8. U 2 OS derived EGFP 2x FYVE expressing cell joe mee ee line only points on the linear portion are shown Doubling time 23 9 hours Cell number natural log 0 T T T T 1 0 25 50 75 100 125 Time hours 25 8010 21UM Chapter 5 Rev B 2006 15 5 2 Assay set up 5 2 1 Live cell EGFP 2x FYVE assay using the IN Cell Analyzer 3000 This manual provides a suggested protocol to use the EGFP 2x FYVE assay for agonist screening on the IN Cell Analyzer 3000 5 2 2 Microplate set up for 96 well format assays The EGFP 2x FYVE assay protocol is optimized for agonist format see sections 5 2 3 It is essential that the number of cells per well in the assay plates be consistent in order to minimize assay variability Wortmannin is used as reference agonist with a typical EC value of 1 88 nM The EGFP 2x FYVE assay can be used with either Hoechst or DRAQ5 as the Nuclear stain As explained in the IN Cell Analyzer 3000 user manual each run must contain a flat field well to compensate for variations in fluorescence intensity across each image It is possible to prepare a plate solely for this purpose Alternatively a designated well on each plate can contain flat field 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 5 2 3 Schematic assay protocol Fig 5 2 shows a typical schematic of an assay to identify PI3K inhibitors
9. all bind to PI 3 P or a similar ligand High affinity binding of FYVE domains to PI 3 P is thought to depend not only on the inositol head group itself but also on presence of the intact lipid within a membranous structure 7 8 FYVE finger proteins can be subdivided into distinct groups comprising proteins with diverse structures and functions While some are involved in membrane tethering others contain kinase phosphatase and GDP GTP exchange factor domains The different groups of FYVE finger proteins participate in distinct cellular processes including vesicle transport cytoskeletal regulation and signal transduction 6 The endosomal pathway is believed to be an important route for the transduction of extracellular stimuli into intracellular responses Fig 1 1 In mammalian cells proteins can be endocytosed via clathrin coated pits the clathrin independent pathway or caveolae within the membrane All of these internalization pathways lead to the appearance of the trafficked protein in early endosomes From early endosomes the protein may be recycled back to the plasma membrane sorted to the late recycling compartment or targeted to late endosomes the pre lysosomal pathway where it is destined for degradation 9 Many FYVE finger proteins have been implicated in membrane trafficking along endosomal lysosomal pathways including EEA1 10 Hrs 11 12 and the yeast proteins Vaclp Vps27p and Fab1p 13 14 Extracellular Recyc
10. 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 should be used For assays in 384 well format please email incellanalyzer uk amersham com for recommendations e ACASY 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 5 CO 95 relative humidity 37 C e mager microscope e g IN Cell Analyzer 3000 or IN Cell Analyzer 1000 e Laminar flow cell culture bench e Tissue culture flasks T flasks and pipettes e Controlled freezing rate device providing a controlled freezing rate of 1 C per minute e Standard tissue culture reagents and facilities section 5 1 1 3 5 IN Cell Analysis System The EGFP 2x FVYE assay has been developed and optimized for analysis using the IN Cell Analyzer 3000 in conjunction with the Granularity Analysis Module Please refer to the instrument user manual for details on instrument set up and the module manual for details on the algorithm settings For further information on either of these products please contact GE Healthcare 3 5 1 IN Cell Analyzer 3000 The IN Cell Analyzer 3000 is a line scanning laser based confocal imaging system with three high speed CCD cameras It has been developed specifically for performing info
11. in the dark whenever practical If a large number of assays are being performed over time during a day we recommend preparing fresh working dilutions at regular intervals with no working solution used once it is 2 hours old e For assays performed on the IN Cell Analyzer 3000 flat field FF solution components are e Cy5 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS e Alexa Fluor 350 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS e Oregon Green 488 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS As explained in the IN Cell Analyzer 3000 user manual prepare the FF solution to give adequate fluorescent signal in each channel used where the fluorescence counts should be less than 3300 at maximum For a Hoechst 33342 nuclear stained assay using ND filters of 0 7 and 1 0 for the 364 and 488 lasers respectively prepare an initial FF solution containing 3 ul 10 uM Oregon Green 488 and 40 ul 100 uM Alexa Fluor 350 in 100 ul PBS For a DRAQS5 nuclear stained assay using ND filters of 1 0 and O for the 488 and 633 lasers respectively use 3 ul 10 UM Oregon Green 488 and 10 ul 10 uM Cy5 in 100 ul PBS Adjust these solutions if required Use 100 ul of FF solution for a 96 well plate and 40 ul FF solution for a 384 well plate 5 1 3 Cell thawing procedure Two cryo vials each containing 1 x 10 cells in 1 ml of Freeze medium are included with this assay kit The vials are stored fro
12. si si2000 20002831 htm Risk assessments made under The Genetically Modified Organisms 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 vapor 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 be used only by responsible persons in authorized areas Care should be taken to prevent ingestion or 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
13. 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 25 8010 21UM Chapter 4 Rev B 2006 11 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 Users of these products MUST make themselves aware of and observe relevant Local Regulations or Codes of Practice For further information refer to the material safety data sheet s and or safety statement s 4 2 Storage The pCORON1000 EGFP 2x FYVE expression vector NIF2022 should be stored at 15 C to 30 C The U 2 OS derived cells expressing the EGFP 2x FYVE fusion protein NIF2021 should be stored at 196 C in liquid Nitrogen 4 3 Handling Upon receipt 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 unless they are required immediately The vector should be removed from the
14. total number of nuclei Ga Fig 5 13 Results obtained using the Granularity Analysis Module for the IN Cell Analyzer 1000 Error SD n 48 20 replicates per data point 25 7 a Control 100 nM Wortmannin Translocation Index granules per cell 25 8010 21UM Chapter 5 Rev B 2006 22 6 Vector use details The plasmid vector pPCORON1000 EGFP 2x FYVE Fig 3 1 can be used to transiently or stably express EGFP 2x FYVE fusion protein in the cell line of choice 6 1 General guidelines for vector use pCORON1000 EGFP 2x FYVE has been used successfully to express EGFP 2x FYVE fusion protein both transiently and stably in the U 2 OS derived cell line Expression levels translocation responses and other assay parameters may vary depending on the cell type and the transfection procedure 6 2 Transient transfection with pCORON1000 EGFP 2x FYVE Transient transfection protocols must be optimized for the cell type of choice Choice of transfection reagent and cell type will affect efficiency of transfection FUGENE 6 Transfection Reagent Roche produced successful results when transfecting pPCORON1000 EGFP 2x FYVE into U 2 OS For more information refer to manufacturer s guidelines for the desired transfection reagent 6 3 Stable cell line generation with OCORON1000 EGFP 2x FYVE The process of establishing stable cell lines involves a large number of variables many of which are cell line dependent Standar
15. 13 c 4425 4720 c 5331 c 5722 6025 c 6231 c 623 4 c 6324 Bcgl 3 1232 2129 5227 c Bfal 11 154 753 1058 1087 2407 2758 3596 3650 5572 5907 6160 Bfrl 4 829 848 1051 3643 Bgll 137 244 366 437 2670 3548 5647 Bglll 1834 6665 Bini 1 3595 Bmyl 19 730 1148 1277 1526 1717 1893 2063 2139 2309 2840 3735 3828 3902 4092 4154 4600 5097 5182 6343 Bpml 3 1552 1792 5696 BpuAl 1 962 BsaAl 3 494 2911 4093 BsaBl 2 2597 4582 BsaHl 8 276 329 412 598 3789 4491 4843 5225 Bsal 2 916 c 5699 BsaJl 19 514 1106 1136 1276 1439 1463 1518 1856 2346 3206 3307 3379 3502 3537 3546 3595 3952 4221 6493 BsaWl 6 1095 1825 3820 5469 6300 6447 BseAl 1 1825 Bsgl 5 1236 c 1333 1657 1954 2200 BsiEl 8 665 1100 2355 2641 3698 5247 5396 6319 BsiHKAI 8 730 1717 3902 4092 4600 5097 5182 6343 BsiVl 24 203 1277 1440 1790 1898 1899 1929 1968 2029 2144 2145 2175 2214 2275 2692 3018 3503 3770 4314 4727 6175 6454 6620 6638 Bsll 24 203 1277 1440 1790 1898 1899 1929 1968 2029 2144 2145 2175 2214 2275 2692 3018 3503 3770 4314 4727 6175 6454 6620 6638 BsmAl 588 826 916 c 941 c 3640 4728 4770 c 4923 c 5699 BsmFl 329 480 648 3289 c 3361 c 3425 c 3940 4472 Bsml 4 1921 2167 2416 2509 c Bsp1286l 19 730 1148 1277 1526 1717 1893 2063 2139 2309 2840 3735 3828 3902 4092 4154 4600 5097 5182 6343 BspDI 2 2601 4570 BspEI 1 1825 BspHI 3 4820 4925 5933 BspMI 4 878 c 3676 c 4057 4507 BspWI 46 137 244 366 398 437 530 554 803 1054 1199 1259 1272 1316 1325 1876 1888 1
16. 192 3960 4038 4119 4128 4206 4583 5082 5118 5135 5393 5439 5457 5798 5903 5915 5993 6001 6012 6087 6665 Dral 4 2556 5187 5879 5898 Drall 1 4785 Dralll 1 2914 Drdl 6 818 2958 3632 3816 4682 6551 Dsal 6 514 1106 1856 3206 3502 4221 DsaV 28 242 435 1135 1151 1276 1388 1463 1517 1789 1967 2213 2345 2346 2605 3307 3362 3379 3791 3951 4174 4691 4726 5227 5578 6274 6492 6505 6626 Eael 11 9 63 1179 1568 2352 3695 3869 4260 4287 4512 5372 Eagl 2 2352 3695 Eam1105l 1 5765 Earl 4 2619 c 4133 c 4343 c 4966 c Ecl136ll 1 728 EcIx 2 2352 3695 Eco47Ill 1 1091 Eco571 7 1261 1305 c 1504 3935 4367 5099 6111 c EcoO1091 1 4785 EcoRI 1 1843 EcoRII 14 242 435 1151 1276 1388 1463 1517 3307 3362 3379 4174 6492 6505 6626 Esp3l 2 4728 4770 c Fnu4Hl 52 835 1267 1326 1348 1632 1639 1690 1693 1787 1880 1926 2074 2126 2172 2320 2352 2355 2480 2662 2694 2708 2730 3201 3543 3698 3750 3761 3851 3856 3893 3934 4021 4024 4027 4263 4359 4400 4414 4515 4625 4734 5021 5250 5345 5372 5711 6039 6245 6248 6313 6456 6611 FnuDll 23 214 1436 1754 1851 2051 2297 2683 2707 2727 3103 3190 3855 4156 4188 4589 4669 4772 4774 4874 5206 5699 6029 6610 Fokl 12 984 c 1135 c 1501 c 2005 c 2251 c 3446 c 4113 4138 4683 c 5326 5613 5794 Fspl 4 2660 3199 3891 5542 Haell 5 1093 2756 2764 3792 6413 Haelll 30 11 65 238 431 1181 1281 1570 1682 2354 2630 2919 3061 3211 3536 3542 3551 3594 3697 3871 4262 4289 4514 4787 5374 5641 5721 6179 6613 6631 6642 Hgal 9 6
17. 2 36 46 43 04 45 7 465 43 02 Fgrains 248 14 260 97 2841 237 37 277 55 254 98 258 12 23107 277 63 250 05 2572 277 73 267 64 286 95 259 64 2727 273 36 245 43 259 46 222 57 273 43 25174 276 65 267 3 221 98 243 22 189 46 253 06 269 59 253 3 242 49 268 54 228 61 254 85 202 52 248 82 253 11 234 1 Is TET iB B F sd 92 23 73 53 8143 87 74 70 21 95 02 93 03 85 58 79 24 88 75 99 35 85 816 31 75 93 87 86 91 30 04 102 84 94 21 103 8 85 71 7266 74 66 82 42 96 69 79 24 104 76 100 59 3174 891 8177 92 03 391 93 68 92 19 96 18 99 67 83 73 18 x To T30 Mean 259 14 2 22 sD 22 25 0 62 d0 u 200 c T amp ir 100 0 oo 300 Time rin Ready NUM Fig 5 4 Data from the example experiment generated by the Granularity Analysis Module exported to and analyzed in Microsoft Excel Further analysis of these results indicated a Z factor of 0 73 for cells treatment with 100 nM Wortmannin imaged at 0 and 30 minutes 5 4 Assay characterization 5 4 1 Translocation index All of the characterizations for the EGFP 2x FYVE assay were performed on the IN Cell Analyzer 3000 using the Granularity Analysis Module The data generated by this module is in the format of Fgrains Fgrains Flux of grains represents a scaled ratio of grain intensity per cell to the total fluorescence inte
18. 2440 2504 3094 3105 3131 3349 3421 3513 5332 5587 5893 Tth1111 1 3907 Xhol 1 1838 Xholl 14 1607 1834 2101 3192 3960 4206 4583 5118 5135 5903 5915 6001 6012 6665 xmal 1 2346 Xmalll 2 2352 3695 Xmnl 3 1998 2244 5165 25 8010 21UM Chapter 11 Rev B 2006 33 GE Healthcare offices GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan GE Healthcare regional office contact numbers Asia Pacific Tel 85 65 6 275 1830 Fax 852 2811 5251 Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 57606 1619 Fax 01 57606 1627 Belgium Tel 0800 73 888 Fax 02 416 82 06 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 France Tel 01 6935 6700 Fax 01 6941 9677 http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK imagination at w
19. 4188 4589 4669 4772 4774 4874 5206 5699 6029 6610 Mwol 46 137 244 366 398 437 530 554 803 1054 1199 1259 1272 1316 1325 1876 1888 1925 2122 2134 2171 2640 2670 2702 2704 2746 2773 2803 3340 3412 3463 3542 3548 3780 3864 3887 4026 4032 4149 4185 4232 4499 4595 5647 6035 6607 6655 Nael 2 2808 4291 Narl 3789 Neil 14 1137 1791 1969 2215 2347 2348 2607 3793 3953 4693 4728 5229 5580 6276 Ncol 6 514 1106 1856 3206 3502 4221 25 8010 21UM Chapter 11 Rev B 2006 31 Enzyme ofcuts Positions c indicates the complementary strand Ndel 1 388 Ndell 31 662 747 1607 1755 1793 1834 2101 2598 2602 2638 3192 3960 4038 4119 4128 4206 4583 5082 5118 5135 5393 5439 5457 5798 5903 5915 5993 6001 6012 6087 6665 NgoMI 2 2806 4289 Nhel 1 1086 Nlalll 32 118 136 458 518 1110 1344 1374 1569 1764 1809 1860 1875 2121 2372 3210 3343 3415 3506 3663 4008 4194 4225 4251 4740 4824 4929 5322 5358 5436 5446 5937 6657 NlalV 21 621979 1145 1683 2103 2839 2851 2872 3247 3313 3385 3790 3825 4585 4878 5468 5679 5720 5814 6586 6625 Notl 1 2352 Nsil 2 3345 3417 Nspl 4 3343 3415 4194 4740 NspV 1 4471 PaeR 7 1 1838 PinAl 1 1095 Plel 11 558 c 836 c 952 c 1068 c 1823 c 2347 2967 2975 c 4454 c 5774 6277 c Ppu10l 2 3341 3413 Psp1406l 2 5163 5536 Pstl 4 839 1930 2176 3842 Pvu 3 665 2641 5396 Pvull 4 2076 2322 3271 3895 Rca 3 4820 4925 5933 Rsa 16 99 373 453 486 537 702 1064 1537 1819 1854 2011 2257 3247 4095 4608 5284 Rsrl 1 4305 Sac 1 730
20. 475 1485 1999 Komada M et al Hrs a tyrosine kinase substrate with a conserved double zinc finger domain is localized to the cytoplasmic surfaces of early endosomes J Biol Chem 272 20538 20544 1997 Schu PV et al Phosphatidyl inositol 3 kinase encoded by yeast VPS34 gene essential for protein sorting Science 260 88 91 1993 Odorizzi G et al Fab1p PI3P 5 kinase function essential for protein sorting in the multivesicular body Cell 95 847 858 1998 Tsukazaki T et al SARA a FYVE domain protein that recruits Smad2 to the TGFb receptor Cell 95 779 791 1998 Komada M and Kitamura N Growth factor induced tyrosine phosphorylation of Hrs a novel 115 kilodalton protein with a structurally conserved putative zinc finger domain Mol Cell Biol 15 6213 6221 1995 Asao H et al Hrs is associated with STAM a signal transducing adaptor molecule J Biol Chem 272 32785 32791 1997 Ponten J and Saksela E Two established in vitro cell lines from human mesenchymal tumours Int J Cancer 2 434 447 1967 Zhang J H et al A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J Biomol Screen 4 67 73 1999 Freshney R Cloning and Selection of Specific Cell Types in Culture of Animal Cells 3rd Edition Wiley Liss Inc Chapter 11 pp 161 178 1994 8010 21UM Chapter 9 Rev B 2006 26 10 Related products Product Name Code GFP Assays GFP PLC8 PH doma
21. 88 1915 2161 2689 4499 4675 5233 5963 c 6541 c HgiAl 8 730 1717 3902 4092 4600 5097 5182 6343 Hhal 34 1092 1397 1438 1754 2051 2091 2297 2661 2685 2698 2707 2729 2755 2763 3200 3783 3791 3855 3892 4158 4188 4190 4418 4671 4774 4874 5206 5543 5636 6029 6138 6312 6412 6479 HinP1I 34 1090 1395 1436 1752 2049 2089 2295 2659 2683 2696 2705 2727 2753 2761 3198 3781 3789 3853 3890 4156 4186 4188 4416 4669 4772 4872 5204 5541 5634 6027 6136 6310 6410 6477 25 8010 21UM Chapter 11 Rev B 2006 30 Enzyme ofcuts Positions c indicates the complementary strand Hincll 678 2343 2495 Hindll 678 2343 2495 Hindlll 757 3611 Hinfl 15 564 842 958 1074 1829 2339 2959 2981 3617 4274 4408 4460 4567 5766 6283 Hpal 1 2495 Hpall 30 1096 1136 1199 1259 1790 1826 1968 2214 2347 2606 2807 3694 3771 3793 3821 3952 4042 4109 4290 4693 4727 5228 5470 5580 5647 5681 6085 6275 6301 6448 Hphl 17 530 1122 1125 c 1455 1479 1608 1956 2202 2911 3967 c 4745 c 4754 c 5038 c 5073 5279 c 5695 5922 Ital 52 835 1267 1326 1348 1632 1639 1690 1693 1787 1880 1926 2074 2126 2172 2320 2352 2355 2480 2662 2694 2708 2730 3201 3543 3698 3750 3761 3851 3856 3893 3934 4021 4024 4027 4263 4359 4400 4414 4515 4625 4734 5021 5250 5345 5372 5711 6039 6245 6248 6313 6456 6611 Kasl 1 3788 Kpnl 3249 Ksp632l 4 2619 c 4133 c 4343 c 4966 c Mael alge 154 753 1058 1087 2407 2758 3596 3650 5572 5907 6160 Maell 20 75 276 288 329 412 493 598 1172 1385 1556 2800 2910 2953 2965 3905
22. 925 2122 2134 2171 2640 2670 2702 2704 2746 2773 2803 3340 3412 3463 3542 3548 3780 3864 3887 4026 4032 4149 4185 4232 4499 4595 5647 6035 6607 6655 BsrBl 4 2767 c 4402 c 4456 4923 c BsrDI 4 66 c 4022 5531 5705 c BsrFl 6 1095 1258 2806 4108 4289 5680 BsrGl 2 97 1817 Bsrl 19 449 c 887 940 1034 c 1719 c 1903 2149 3000 3480 c 3733 3934 5120 5290 c 5559 5602 5720 6126 6238 c 6251 c BssHII 1 4186 BstBl 4471 BstNI 14 244 437 1153 1278 1390 1465 1519 3309 3364 3381 4176 6494 6507 6628 25 8010 21UM Chapter 11 Rev B 2006 29 Enzyme ofcuts Positions c indicates the complementary strand BstUl 23 214 1436 1754 1851 2051 2297 2683 2707 2727 3103 3190 3855 4156 4188 4589 4669 4772 4774 4874 5206 5699 6029 6610 BstX 1 4510 BstY 14 1607 1834 2101 3192 3960 4206 4583 5118 5135 5903 5915 6001 6012 6665 Cfol 34 1092 1397 1438 1754 2051 2091 2297 2661 2685 2698 2707 2729 2755 2763 3200 3783 3791 3855 3892 4158 4188 4190 4418 4671 4774 4874 5206 5543 5636 6029 6138 6312 6412 6479 Cfr10l 6 1095 1258 2806 4108 4289 5680 Clal 2 2601 4570 Csp45l 1 4471 Csp6l 16 98 372 452 485 536 701 1063 1536 1818 1853 2010 2256 3246 4094 4607 5283 Dadel 14 1711 1729 1831 1910 2156 3253 3555 4452 4603 4838 5264 5804 5970 6379 Dpnl 31 664 749 1609 1757 1795 1836 2103 2600 2604 2640 3194 3962 4040 4121 4130 4208 4585 5084 5120 5137 5395 5441 5459 5800 5905 5917 5995 6003 6014 6089 6667 Dpnll 31 662 747 1607 1755 1793 1834 2101 2598 2602 2638 3
23. Chapter 3 Rev B 2006 10 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 All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In the case of contact with skin or eyes wash immediately with water 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 Contained Use Regulations 2000 Information to assist users in producing their own risk assessments is provided in sections 3 3 1 and 3 3 2 of The Genetically Modified Organisms contained use Regulations 2000 http wwwlegislation hmso gov uk
24. GE Healthcare EGFP 2x FYVE Assay Product User Manual Codes 25 8010 21 25 8010 22 25 8010 23 25 8010 24 Page finder 1 Introduction 1 1 FYVE domains as cellular sensors of phosphoinositol 3 phosphate 1 2 The FYVE finger 1 3 EGFP 2x FYVE assay 2 Licensing considerations 2 1 Right to use 2 2 Legal 3 Product contents 3 1 Component summary 3 2 U 2 OS derived cell line expressing EGFP 2x FYVE fusion protein NIF2021 3 2 1 U 2 OS derived parental cell line 3 2 2 U 2 OS derived EGFP 2x FYVE expressing cell line 3 3 EGFP 2x FYVE expression vector NIF2022 3 4 Materials and equipment required 3 5 IN Cell Analysis System 3 5 1 IN Cell Analyzer 3000 3 5 2 Granularity Analysis Module 3 5 3 IN Cell Analyzer 1000 3 6 EGFP 2x FYVE translocation assay on epifluorescence microscopes 3 7 Software requirements 4 Safety warnings handling and precautions 4 1 Safety warnings 4 2 Storage 4 3 Handling 4 3 1 Vector 4 3 2 Cells 5 Cell assay design 5 1 Culture and maintenance of U 2 OS derived EGFP 2x FYVE expressing cell line 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 freezing procedure 5 1 7 Growth characteristics 5 2 Assay set up 5 2 1 Live cell EGFP 2x FYVE assay using the IN Cell Analyzer 3000 5 2 2 Microplate set up for 96 well
25. and mammalian cells EMBO Journal 19 4577 4588 2000 Vanhaesebroeck B and Waterfield MD Signalling by distinct classes of phophoinositide 3 kinases Experimental Cell Research 253 239 254 1999 Pattni K Jepson M Stenmark H Banting G A PtdIns 3 P specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells Current Biology 11 1636 1642 2001 Stein RC and Waterfield MD PI3 kinase inhibition a target for drug development Molecular Medicine Today 6 347 358 2000 Stenmark H et al Endosomal localization of the autoantigen EEA1 is mediated by a zinc binding FYVE finger J Biol Chem 271 24048 24054 1996 Kutateladze T Overduin M Structural mechanism of endosome docking by the FYVE domain Science 291 1793 1796 2001 Cullen PJ Cozier GE Banting G Mellor H Modular phosphoinositid binding domains their role in signalling and membrane trafficking Current Biology 11 R882 R893 2001 Sorkin A The endocytosis machinery J Cell Sci 113 Pt24 4375 4376 2000 J Mu FT et al EEA1 an early endosome associated protein EEA1 is a conserved alpha helical peripheral membrane protein flanked by cysteine fingers and contains a calmodulin binding IQ motif J Biol Chem 270 13503 13511 1995 Komada M and Soriano P Hrs a FYVE finger protein localized to early endosomes is implicated in vesicular traffic and required for ventral folding morphogenesis Genes and Development 13 1
26. ark of Microsoft Corporation Hoechst is a trademark of Aventis Geneticin is a registered trademark of Life Technologies Inc DRAQS is a trademark of Biostatus Limited 2006 General Electric Company All rights reserved GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specification and features shown herein or discontinue the product described at any time without notice or obligation Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 25 8010 21UM Chapter 2 Rev B 2006 7 3 Product contents 5 1 Component summary U 2 OS derived cells expressing the EGFP 2x FYVE fusion protein two vials each containing 1 ml and 1 x 108 cells NIF2021 e pCORON1000 EGFP 2x FYVE expression vector one vial containing 10 ug DNA at a concentration of 250 ug ml supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 0 NIF2022 e User manual 3 2 U 2 OS derived cell line expressing EGFP 2x FYVE fusion protein NIF2021 3 2 1 U 2 OS derived parental cell line The parental cell line U 2 OS ATCC HTB 96 was derived from a moderately differentiated sarcoma of the tibia of a 15 year old girl 18 The U 2 OS cell line is choromosomally highly alerted with chromosome counts in the hypertrip
27. ch 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 8 1 2 Verify density of cell plating adjust plating density to values that yield optimal assay response 8 1 3 Check that your primary parameters are correct and suitable for the cells currently in use 8 1 4 Ensure that proper incubation is maintained as consistently as possible during the assay When the plate are out of the CO incubator for extended periods it is essential that HEPES buffer be added to the medium to maintain the correct pH 8 1 5 Repeat assay with fresh reagents 8 1 6 Use activly growing cells maintained at 37 C Pre warm reagents to 37 C 8 2 1 Adjust nuclear stain concentration to recommended level 8 2 2 Adjust nuclear stain incubation time to recommended length 8 3 1 Alignment and calibration of instrument Perform Z stack on cells Change autofocus offset 8 4 1 Reduce plating density 8 5 1 Apply flatfield correction or adjust flatfield solution 9 References 10 11 12 13 14 15 16 17 18 19 20 25 Stenmark H and Aasland R FYVE finger proteins effectors of an inositol lipid Journal of Cell Science 112 4175 4183 1999 Gillooly DJ Morrow I Lindsay M Gould R Bryant NJ Gaullier J M Parton RG and Stenmark H Localization of phosphatidylinositol 3 phosphate in yeast
28. cryo porter and stored at 15 C to 30 C until required 4 3 1 Vector After thawing the DNA sample centrifuge briefly to recover the contents 4 3 2 Cells Do not centrifuge the cell samples upon thawing 25 8010 21UM Chapter 4 Rev B 2006 12 5 Cell assay design 5 1 Culture and maintenance of U 2 OS derived EGFP 2x FYVE expressing 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 and to perform the assay GIBCO Dulbecco s Modified Eagle Media DMEM with Glutamax 1 Invitrogen life technologies 31966 021 or equivalent Fetal Bovine Serum FBS JRH Biosciences 12103 or equivalent GIBCO Penicillin Streptomycin P S 5000 units ml Penicillin G Sodium and 5000 ug ml Streptomycin Sulfate Invitrogen life technologies 15140 122 or equivalent Geneticin G418 Sigma G 7034 or equivalent GIBCO Trypsin EDTA in HBSS w o Calcium or Magnesium Invitrogen life technologies 25300 054 or equivalent GIBCO HEPES Buffer 1 M solution Invitrogen life technologies 15630 056 or equivalent Bovine Serum Albumin BSA Sigma A 7888 or equivalent GIBCO Phosphate Buffered Saline PBS Dulbecco s w o Calcium Magnesium or Sodium Bicarbonate Invitrogen life technologies 14190 094 or equivalent Dimethylsulfoxide DMSO Sigma D 5879 or equivalent GIBCO Nutrient Mixture F 12 Ham medium with Glutamax Invitrogen life technolo
29. ctral overlap between DRAQ5 and GFP images can be taken simultaneously Fig 5 11 shows a Wortmannin dose response curve where the nuclear marker has been changed to 1 uM DRAQS 150 5 Fig 5 11 Wortmannin dose response curve using 1 mM DRAQS5 as the 3 nuclear marker Error SD n 8 lt a 1004 replicates per data point E 26 EC 1 64 nM OD ee 2 5 504 E 0 7 T T T 1 11 10 9 8 7 6 Wortmannin M 5 4 10 Results obtained on the IN Cell Analyzer 1000 The following figures Fig 5 12 and Fig 5 13 were generated from a single experiment and provide an example of the images and results that can be obtained with the EGFP 2x FYVE assay using the IN Cell Analyzer 1000 Assays were performed as described in section 5 2 except that after the 30 minute incubation the cells were fixed using 2 formaldehyde Fig 5 12 shows images captured on the IN Cell Analyzer 1000 using a 20X air objective lens EGFP 2x FYVE expressing cells were fixed 30 minutes after the addition of assay buffer control cells or 100 nM wortmannin a ib Fig 5 12 Images taken on the IN Cell Analyzer 1000 of EGFP 2x FYVE expressing cells Cells were fixed using 2 Formaldehyde 30 minutes after the addition of a assay buffer control and b 100 nM Wortmannin Fig 5 13 shows a graphical representation of the translocation The translocation index is defined as Granule Count Cell This is the total number of granules per image divided by the
30. d methods and guidelines for the generation of stable cell lines are widely available in the public domain 20 pCORON1000 EGFP 2x FYVE has been used to generate stably transfected cell populations The magnitude of the response and the kinetics of the translocation event achievable with different cell lines are unknown and may deviate from the values specified in this manual 25 8010 21UM Chapter 6 Rev B 2006 23 7 Quality control 7 1 EGFP 2x FYVE Cell Line The EGFP 2x FYVE cell line is supplied at a concentration of 1 x 108 cells ml in fetal calf serum containing 10 DMSO The cell line has the characteristics detailed in Table 7 1 Property Value Measurement method Assay stability Z factor gt 0 6 Quality Control Assay Viability from frozen 80 CASY1 Model TT Cell Counter Cell diameter um 18 21 CASY1 Model TT Cell Counter Fluorescence at 5 x 104 gt 28 000 for 20 FARCyte Gain 62 cells ml RFU passages after dispatch 7 2 EGFP 2x FYVE expression vector The EGFP 2x FYVE expression vector is supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 9 at 250 ug ml The vector has the characteristics outlined in Table 7 2 Property Value Limits Measurement method Concentration 250 ug ml UV Absorbance 260 nm in water Purity Minimal A260 Azg0 ratio Between 1 8 2 2 UV Vis Absorbance contamination of 260 nm and 280 nm the DNA construct by RNA or protein Expected restriction The restriction Agarose gel pattern digest
31. d objects 25 8010 21UM Chapter 3 Rev B 2006 9 3 6 EGFP 2x FYVE translocation assay on epifluorescence microscopes For speed of screening and quality of the images obtained we recommend performing the EGFP 2x FYVE assay on the IN Cell Analyzer platforms However it is possible to adapt the assay to be read 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 40 x 1 3 NA or similar and epifluoresence filter sets compatible with GFP and the desired nuclear dye 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 analysis 3 7 Software requirements IN Cell Analyzer 3000 and IN Cell Analyzer 1000 The Granularity Analysis Modules are available from GE Healthcare for automated image analysis of the EGFP 2x FYVE assay Analyzed data are exported as numerical files in ASCII format ASCII format data can be imported into Microsoft Excel Microsoft Access or any similar package for further data analysis as desired Confocal or epifluorescence microscope Suitable software will be required for analysis of images acquired on microscopes other than the IN Cell Analyzer Systems 25 8010 21UM
32. ent Signal to Noise 1312422 15 48 operators on different occasions using the suggested protocol Z factor 0 76 0 04 15 48 SD shown is the standard deviation of Magnitude of Response 229 61 17 50 15 48 the assays CV Signal to noise is mean signal of j control mean signal of Wortmannin Stimulated 38 08 6 62 15 48 control standard deviation 19 Unstimulated 7 61 1 22 15 48 Magnitude of response is the mean background control mean signal with Wortmannin CV is standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of 5 4 3 Seeding density assay quality i9 Fig 5 5 shows the effect of varying seeding density in a 96 well microplate The data were collected 30 minutes after the addition of 100 nM Wortmannin Significant differences between stimulated and non stimulated cells were seen at cell densities ranging from 0 2 x 104 to 1 2 x 10 cells per well We recommend seeding in the range 0 4 x 104 to 1 x 104 cells per well 300 5 h Ea x Fig 5 5 Wortmannin induced EGFP E Control 2x FYVE translocation as a function cp auo HEE Wortmannin of seeding density Cells were treated 5 with 100 nM Wortmannin for 2 P 30 minutes prior to the imaging Error w 1004 SD n 8 replicates per data point O 0 ae ro SS emn ae a a S S 3 S a S x x x x x x N a Ne ioe fo N oO oO Cn d
33. essing cell imaged 30 minutes after treatment with 100 nM Wortmannin Bioimage is a Danish Biotech company specializing in developing drug candidates that exert their activity through modulation of protein translocation For more information visit their Web site at www bioimage dk 1 Introduction 1 1 FYVE domains as cellular sensors of phosphoinositol 3 phosphate The FYVE finger is a cysteine rich domain of approximately 60 80 amino acids that binds specifically to the inositol head group of phosphatidylinositol 3 phosphate PI 3 P For review see 1 PI 3 P has been implicated as an important mediator of vesicular transport and is abundant in early endosome membranes and internal membrane structures of late multivesicular endosomes MVBs 2 The majority of cellular PI 3 P is thought to be generated by the constitutive action of a class Ill phosphatidylinositol 3 kinases PI3 kinase 3 PI3 kinases phosphorylate phosphatidylinositol at the 3 position of the inositol ring to produce PI 3 P and in so doing help to maintain PI 3 P levels in the membrane Inhibitors of PI3 kinase activity induce a decrease in PI 3 P levels observed in endosomal MVB membranes 2 A GFP reporter molecule based on tandem FYVE domains from the human homologue of hepatocyte growth factor regulated tyrosine kinase substrate Hrs has been shown in live cells to localize to PI 3 P on early endosome membranes in a PI3 kinase dependent fashion and may be expl
34. format assays 5 2 3 Schematic agonist assay protocol 5 2 4 Assay protocol 96 well format 5 3 Results 5 3 1 Calculating Z factor 5 3 2 Example results 25 8010 21UM Pagefinder Rev B 2006 o NOD WNW o OOO O O 10 10 11 11 12 12 12 12 13 13 13 13 14 14 15 15 15 16 5 4 Assay characterization 18 5 4 1 Transloction index 18 5 4 2 Summary of quantitive assay parameters 18 5 4 3 Seeding density 19 5 4 4 Wortmannin dose response 19 5 4 5 Time course 20 5 4 6 Sensitivity of assay to DMSO Ethanol and Methanol 20 5 4 7 Effect of different assay media 21 5 4 8 Effect of serum starvation 21 5 4 9 Effect of using the nuclear marker DRAQ5 on the Translocation 22 5 4 10 Results obtained on the IN Cell Analyzer 1000 22 6 Vector use details 23 6 1 General guidelines for vector use 23 6 2 Transient transfection with pCORON1000 EGFP 2x FYVE 23 6 3 Stable cell line generation with pCORON1000 EGFP 2x FYVE 23 7 Quality control 24 7 1 EGFP 2x FYVE cell line 24 7 2 EGFP 2x FYVE expression vector 24 8 Troubleshooting guide 25 9 References 26 10 Related products 27 11 Appendices 28 11 1 Appendix A Restriction map of pCORON1000 EGFP 2x FYVE 28 Front cover Top image U 2 OS derived EGFP 2x FYVE expressing cell line before the addition of Wortmannin Hoechst nuclear stain is also shown Imaged on the IN Cell Analyzer 3000 Bottom image U 2 OS derived EGFP 2x FYVE expr
35. gies 31765 027 or equivalent Wortmannin Sigma W 1628 or equivalent Hoechst 33342 Trihydrochloride fluoropure grade Molecular Probes H 21492 DRAQ5 Biostatus Cy5 monocarboxyl dye GE Healthcare PAO5111 Oregon Green 2 7 difluorofluorescein Molecular Probes D 6145 Alexa Fluor Carboxylic acid Succinimidyl Ester Molecular Probes A 10168 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 Growth medium DMEM with Glutamax 1 supplemented with 10 v v FBS 1 v v Penicillin Streptomycin and 0 5 mg ml Geneticin Freeze medium DMEM with Glutamax 1 supplemented with 10 v v FBS 1 v v Penicillin Streptomycin and 10 v v DMSO Assay medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 mM HEPES 0 1 w v BSA and 1 0 uM Hoechst Nuclear stain Wortmannin Wortmannin is light sensitive Care must be taken in handling to prevent excessive degradation Add 5 mg Wortmannin to 0 5 ml DMSO Make up to 100 ml using PBS to give a stock of 117 uM This should be kept in the dark at 4 C throughout the assay whenever possible Prepare a 400 nM working dilution with Assay medium four fold of the final concentration less than an 25 8010 21UM Chapter 5 Rev B 2006 13 hour before it is required on the day of the assay keeping this solution
36. in assay See below GFP Rac1 assay See below GFP MAPKAP k2 assay See below AKT1 EGFP assay See below Use of the GFP assays is limited as stated in the terms and conditions of sale The product codes vary accordingly Please contact your local representative for details CypHer pCORON1000 VSV G tag Expression vector 25 8008 51 pCORON1000 SP VSV G tag Expression vector 25 8009 92 CypHerS labeled anti VSV G antibody PA45407 CypHer5 NHS ester 1 mg pack PA15401 CypHer5 NHS ester 5 mg pack PA15405 IN Cell Analysis System IN Cell Analyzer 3000 25 8010 11 Granularity Analysis Module for IN Cell Analyzer 3000 63 0048 97 IN Cell Analyzer 1000 25 8010 26 Granularity Analysis Module for IN Cell Analyzer 1000 25 8010 30 25 8010 21UM Chapter 10 Rev B 2006 27 11 Appendices 11 1 Appendix A Restriction map of pPCORON1000 EGFP 2x FYVE The following enzymes do not cut the vector Apal Ascl BbrPI Bcll Bou1102I BsiWl Bsp120I Bst11071 BstEll Bsu36l Celll EcoNI EcoRV Espl Kspl Nrul Pacl PfIMI PmaCl Pmel Pmil PouMI Sacll SgrAl Swal Van91l Xbal Xcml Enzyme ofcuts Positions c indicates the complementary strand Aatl 1 3594 Aatll 5 279 332 415 601 4846 Acc65l 1 3245 Accl 1 2342 Acclll 1 1825 Acil 79 129 212 240 252 266 399 433 524 c 557 c 669 690 c 767 c 1326 1367 1434 1473 1611 1724 1784 1787 1923 2169 2351 c 2355 2632 2693 c 2707 c 2710 c 2738 2765 3143 c 3169 c 3182 3190 c
37. l for T 162 flasks and gently resuspend the cells with a 10 ml pipette until all the clumps have dispersed 7 Aspirate the cell suspension and dispense 2 ml cells into a new culture vessel 25 8010 21UM Chapter 5 Rev B 2006 14 5 1 5 Cell seeding procedure The following procedure is optimized for cells grown in standard T 75 and T 162 flasks to be seeded into 96 well microplates 1 Warm all reagents to 37 C 2 Aspirate the medium from the cells and discard 3 Wash the cells with PBS Take care not to damage the cell layer while washing but ensure that the entire cell surface is washed 4 Aspirate the PBS from the cells and discard 5 Add Trypsin EDTA 2 ml for T 75 and 4 ml for T 162 flasks ensuring that all cells are in contact with the solution Wait for 3 10 minutes for the cells to round up loosen Check on an inverted microscope 6 When the cells are loose tap the flask gently to dislodge the cells Add Growth medium 3 ml for T 75 and 6 ml for T 162 flasks and gently resuspend the cells with a 10 ml pipette until all the clumps have dispersed 7 Count the cells using either a CASY1 Cell Counter and Analyzer System Model TT or a hemocytometer 8 Using fresh Growth medium adjust the cell density to deliver the desired number of cells to each well For example to add 0 6 x 104 cells per well in a volume of 200 ul adjust the suspension to 3 x 104 cells per ml We recommend a concentration of 2 5 x 10 cell
38. ling endosome Late endosome Early Primary endosome endosome Orange Rabbd binding domain FYVE PI3P binding domain EEA1 HRS Blue Ubiquitin interacting domain Red Clathrin binding domain Lysosome Fig 1 1 The role of FYVE finger proteins in vesicle transport provided with permission from BioCarta www biocarta com 25 8010 21UM Chapter 1 Rev B 2006 4 Although membrane trafficking is the most well characterized role proposed for FYVE finger proteins some FYVE domain containing proteins appear to have alternative functions Several lines of evidence suggest that the FYVE domain may play a role in localizing signalling components to particular intracellular sites For example SARA Smad anchor for receptor activation contains a FYVE domain that plays a critical role in transforming growth factor B TGFB induced signal transduction SARA is believed to mediate recruitment of Smad 2 and Smad 3 to intracellular membranes most likely endosomes containing internalized TGFR receptor 15 The activated receptor kinase facilitates phosphorylation dependent interaction of either Smad2 or Smad 3 proteins with Smad 4 The resulting complex then translocates to the nucleus where it activates transcription of target genes Hrs has also been implicated in signal transduction due to observations that it undergoes agonist dependent phosphorylation 12 16 and associates with signal transducing adaptor molecule STAM a media
39. loid range and expresses the insulin like growth factor and II receptors 3 2 2 U 2 OS derived EGFP 2x FYVE expressing cell line U 2 OS cells were transfected with the pCORON1000 EGFP 2x FYVE vector supplied using the FUGENE 6 transfection method according to the manufacturer s instructions A stable clone expressing the recombinant fusion protein was selected using 500 ug ml Geneticin for approximately two weeks The isolated clone was grown for 4 passages before freezing The cells tested negative for mycoplasma bacterial and yeast contamination testing details are available upon request 3 3 EGFP 2x FYVE expression vector NIF2022 The 6 7 kb plasmid PCORON1000 EGFP 2x FYVE contains a bacterial ampicillin resistance gene and a mammalian neomycin resistance gene see Fig 3 1 The sequence of the construct is available on a CD upon request Please e mail incellanalyzer uk amershambiosciences com A detailed restriction map is shown in chapter 11 appendix A CMV enhancer Fig 3 1 Vector map of the supplied EGFP 2x FYVE expression vector vy CMV promoter S Chimeric intron Ampicillin resistance gene pCORON1000 EGFP 2xFYVE 6669 bp EGFP 2xFYVE Mlul 1849 lt BamHI 4583 fg BamHI 2101 Synthetic poly A sall 2341 Neomycin resistance gene a SV40 late polyA f1 ori SV40 enhancer early promoter 25 8010 21UM Chapter 3 Rev B 2006 8 3 4 Materials and equipment required The following materials
40. lumbia University under US patent numbers 5491084 and 6146826 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 and University of Florida Research Foundation under patents US 5968750 5874304 5795737 6020192 and other pending and foreign patent applications and lowa Research Foundation The CMV promoter is covered under US patents 5168062 and 5385839 and its use is permitted for research purposes only Any other use of the CMV promoter requires a license from the University of owa Research Foundation 214 Technology Innovation Center lowa City A52242 USA and Cedars Sinai Medical Centre For the FYVE domain under US patent 6376174B1 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 25 8010 21UM Chapter 2 Rev B 2006 6 2 2 Legal GE and GE monogram are trademarks of General Electric Company Cy is a trademark of GE Healthcare companies Biolmage and Redistribution are trademarks of Biolmage A S Biocarta is a trademark of Biocarta Inc FUGENE is a trademark of Fugent LLC Microsoft is a tradem
41. n wo 2702 144 95 95 100 26 33 198 ns 15 100 24 08 146 36 36 100 24 08 48 100 100 100 2578 2n ne ne 100 25 86 n n2 n2 100 25 23 154 34 34 100 2348 193 E a1 100 238 184 123 123 100 25 58 158 EJI EJ 100 22 14 184 137 187 100 24m 192 7 7 100 28 155 95 35 100 24 38 207 113 113 100 25 25 178 106 106 100 2824 161 108 108 100 22 81 ne 83 83 100 21 64 202 ne ne 100 2383 178 74 74 100 19 12 161 94 oF 100 a 159 107 107 100 22 69 216 137 137 100 23 36 207 122 122 100 2108 185 127 127 100 24 03 163 108 108 100 20 08 158 101 101 100 24 1 187 a1 at wo 13 35 73 ng ne 100 25 13 182 121 121 100 26 21 193 m 14 100 2739 id 41 gt bih wort time 5 min 2h_Population_0 Draw Ts G AutoShapes a O BA L A E N sd Agrains 10 25 92 83 ni 105 16 10 32 112 29 9 82 18 8 64 109 35 10 97 939 68 9 32 96 53 10 11 12 5 9 32 11 36 10 24 101 57 10 92 106 33 9 22 106 7 9 32 10 4 10 11 107 23 10 17 100 08 78 103 67 22 108 7 1 23 94 91 11 33 103 52 11 66 93 88 9 33 103 21 8 83 106 67 10 39 16 44 845 938 07 9 82 88 42 9 72 103 24 1 65 82 2 8 67 88 47 10 09 97 18 10 43 99 87 88 89 18 3 89 102 2 97 84 83 308 99 61 10 39 83 11 12 16 104 96 14 106 77 10 71 17 73 ea A sd 39 35 41 33 437 404 34 85 40 93 39 7 40 09 38 16 44 83 46 14 36 18 38 29 39 09 478 32 22 46 34 45 52 46 29 46 68 36 92 35 51 38 91 33 08 39 85 33 1 49 67 36 99 40 57 42 07 36 3 38 53 38 6
42. nsity in the cell measurement region Data plots throughout this manual are based on the population averaged Fgrain value obtained from all cells imaged in individual sample wells This translocation index is used in all data presented in sections 5 4 2 5 4 9 5 4 2 Summary of quantitative assay parameters A summary of typical assay data using Wortmannin as a PI3K inhibitor is shown in Tables 5 1 and 5 2 Table 5 1 shows the results obtained from a single assay plate indicating the level of well to well variation Table 5 2 shows a summary of the results obtained from 15 assays performed by different operators on different occasions giving an indication of inter assay variation Table 5 1 Results from a typical single assay performed using the suggested protocol Signal to noise is mean signal of control mean signal of Wortmannin control standard deviation 19 Magnitude of response is the mean background control mean signal with Wortmannin Parameter Assay Data Assays Replicates Signal to Noise 12 42 1 48 Z factor 0 75 1 48 Magnitude of Response 219 82 1 48 CV Stimulated 32 67 1 48 Unstimulated 7 99 1 48 25 8010 21UM Chapter 5 Rev B 2006 18 CV is standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of assay quality 19 Parameter Assay Data Assays Replicates Table 5 2 Summary results from SD assays performed by differ
43. oited as an endosomal marker and reporter for the presence of cellular PI 3 P 2 4 Since the majority of PI 3 P is maintained through class III PI3 kinase activity FYVE domain reporter proteins may also potentially be used in conjunction with other phosophoinositide reporters as class Ill specific PI3 kinase sensors although the class specificity of the EGFP 2X FYVE reporter remains to be confirmed P 3 kinases play critical roles in the regulation of many cellular processes including cell proliferation survival motility and metabolism and are therefore of great interest as therapeutic targets for review see 5 Three main classes of P 3 kinase have been identified to date based on structure binding partners mode of activation and in vitro substrate specificity Class PI3 kinases are activated by extra cellular signalling through receptor tyrosine kinases and some GPCRs The phoshoinositide products of class PI3 kinases are often binding substrates for pleckstrin homology PH domain containing proteins such as Akt1 PDK1 and Tec kinases Class PI3 kinases have been implicated in oncogenesis and tumor progression and have therefore recently attracted attention as potential targets for cancer therapy Class II PI3 kinases are the least understood class but their subcellular localization has implicated them in vesicle formation or sorting events Class III PI3 kinases which are homologues of the yeast vacuolar sorting protein vps27 a
44. on the IN Cell Analyzer 3000 of the supplied U 2 OS derived EGFP 2x FVYE expressing cells before and after treatment with 100 nM Wortmannin Following image analysis the population data is exported into Microsoft Excel for further manipulation Fig 5 4 Fig 5 3 The same cells expressing EGFP 2x FYVE a before and b 30 minutes after treatment with 100 nM Wortmannin 25 8010 21UM_ Chapter 5 Rev B 2006 17 s Eile Edit Yiew Insert Format Tools Data Window Help Ooh SRY tad Arial 0 B Zz u s20 x E AE e EE Plate Cyole 41 uzos 42 u20s _43 u208 44 u20s 45 u20s 46 u20s 47 u20s 48 uz0s 43 uz0s 50 u20s 51 u2o0s 52 uzos 53 uz0s 54 u20s 55 u20s 56 uz0s 5 u20s 58 U20S 59 U20S 60 u20s 61 u20s 62 u20s 63 u20s 64 uz0s 65 u20s 68 u20s 67 uz0s 68 u20s 63 u20s 70 u20s 71 u20s 72 uz0s 73 u20s 74 uz0s 75 u20s 76 u20s 7 uz0s 78 uz0s well oa 087 oc 0D 0 E OF 0 G 0 H 0 A8 0 B68 0 c8 0 D8 0 E8 OFS 0 Gs 0 H8 049 0 B3 0 c3 0 D3 0 E9 OFS 0 G9 0 H3 0 A10 0 B10 0 cio 0 D10 0 E10 0 F10 0 G10 0 H10 0 AN 0 Bt oc oon o En 0 Fi Msg sooooooocooooooooooooooooooooooooooooo0oo 0 0 00 Ntot Npass Npos pos__Ngrains 165 140 140 100 22 31 160 105 105 100 25 07 159 101 101 100 2564 148 37 a7 100 2205 129 73 73 100 25 59 154 83 83 100 24 89 195 78 78 wo 2221 179 119
45. on the translocation of EGFP 250 4 I I 7 i 2x FYVE Error SD n 4 replicates y G 2007 I per data point a 1504 of 505 oe OD ow ge Cc 2 E k a a a a kw a a a 0 S 2 3 amp 8 a ag amp Yi ao a a m a g a a o rt g BF 8F B x xX E 3 N FB HY Q aN ic fo e So I E g 2 T 3 3 2 Q gg gt z 3 a D T T Oo oe a a oO v T x x SS T 2 S u 2 E Egg ie Q S r nN o a nN Ta i S 4 a u i T i A ono n 5 E 2 6 ie io T jo ac 5 4 8 Effect of serum starvation To determine the effect of serum starving the cells prior to the addition of Wortmannin cells were incubated in Assay media for 0 4 hours The results shown in Fig 5 10 demonstrate that serum starvation is not necessary prior to assaying the Wortmannin stimulated translocation a Control Fig 5 10 The effect of serum Wortmannin starvation on the translocation of EGFP 2x FYVE Error SD n 4 300 z f replicates per data point S 2504 E o 200 5 150 g 2 507 2 jo o4 z 7 y i 1 2 3 4 5 Starvation Time hours 25 8010 21UM Chapter 5 Rev B 2006 21 5 4 9 Effect of using the nuclear marker DRAQ5 on the translocation GFP expressing cells which have been stained using Hoechst nuclear marker must be imaged sequentially due to overlaping spectral profiles of these two probes For speed of imaging the red nuclear marker DRAQS5 can be used instead of Hoechst Since there is no spe
46. ork Germany Tel 089 96281 660 Fax 089 96281 620 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 813 5331 9336 Fax 813 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0800 8282821 Fax 0800 8282824 Norway Tel 815 65 555 Fax 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 Tel 7 495 956 5177 Fax 7 495 956 5176 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Russia amp other C 1 S amp N I S Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 25 8010 21UM Rev B 2006
47. re believed to be important for intracellular membrane trafficking but have also been shown to be involved in regulation of key extracellular signaling pathways such as the TGFB pathway that signal through the endosome Class III PI3 kinase pathways are typically mediated by FYVE domain containing proteins including EEA1 p235 Hrs SARA and Fgd1 Because P 3 kinases are involved in diverse critical signalling and trafficking pathways inhibitors that act on multiple classes are likely to have unacceptably high degrees of toxicity and non specific effects 5 Most PI3 kinase inhibitors identified to date act to some extent on more than one PI3 kinase class in vitro PI3 kinase sensors such as the EGFP 2X FYVE domain may be useful in the discovery and development of more selective therapeutic inhibitors 1 2 The FYVE finger The FYVE domain was named after four proteins in which it was originally found namely Fab1 YOTB Vac1p and EEA1 FYVE domains have eight conserved cysteine residues that co ordinate two Zn ions in a specific conformation The third cysteine residue lies within a highly conserved basic motif R R KJHHCRxCG motif that mediates binding of the inositol head group of PI 3 P 6 The biochemical function of the FYVE domain was uncovered when it was discovered that several FYVE finger proteins specifically bind PI 3 P The highly conserved nature of FYVE 25 8010 21UM Chapter 1 Rev B 2006 3 fingers strongly suggests that they
48. rmation rich cellular assays very rapidly and at high resolution enabling high throughput and high content testing of drug compounds 3 5 2 Granularity Analysis Module The Granularity Analysis Module is used to measure the degree of granularity within the cell Granular fluorescence is defined as focal regions within the cell having a defined intensity difference compared to their background This speckled appearance is often due to accumulation of the fluorophore into discrete subcellular compartments and thus the number size and intensity of the granules can be used as an indicator of compartmentalization 3 5 3 IN Cell Analyzer 1000 The IN Cell Analyzer 1000 is a bench top automated microscope system designed for imaging sub cellular end point assays The system s core components are a Nikon microscope xenon lamp high resolution CCD camera variable objective and filter choices laser auto focus and motorized stage Additional optional modules include liquid handling and temperature control to enable imaging of live cell assays There are a number of analysis modules available with the system as well as the capability to export images and data into other commercial analysis packages The Granularity Analysis Module for the IN Cell Analyzer 1000 quantifies images with respect to granule count area and intensity in relation to size scales The term granule as used here is not limited to spherical forms and includes irregularly shape
49. s per ml 9 Dispense 200 ul of the cells into each well of the microplate except the well reserved for the flat field solution see IN Cell Analyzer 3000 manual for further information 10 Incubate the plated cells for 24 hours at 37 C before starting the assay N B If the cells are near confluence prior to trypsinization they should be split into two T flasks They will then be ready for seeding the following day 5 1 6 Cell freezing procedure 1 Harvest the cells as described in section 5 1 4 and resuspend the cells in a small volume of Growth medium 2 Count the cells as described in section 5 1 5 3 Pellet the cells at approximately 300 g for 5 minutes Aspirate the medium from the cells 4 Gently resuspend the cells until no clumps remain in Freeze medium at a concentration of 1 x 10 cells in 1 ml and transfer into cryo vials Each vial should contain 1 x 108 cells in 1 ml of Freeze medium 5 Transfer the vials to a cryo freezing device and freeze at 80 C for 16 24 hours 6 Transfer the vials to the vapor phase in a liquid Nitrogen storage device 5 1 7 Growth characteristics Under standard growth conditions the cells should maintain an average size of 20 5 um as measured using a CASY1 Cell Counter and Analyzer System Model TT The doubling time of the cell line in exponential growth phase has been determined to be approximately 24 hours under standard conditions Fig 5 1 Fig 5 1 Growth curve of the
50. s should electrophoresis give fragments of the sizes shown in Table 7 3 Enzyme s of cuts Fragment s size bp Ncol 6 296 592 719 750 1350 2962 Pvull 4 246 624 949 4850 BamHI 2 2482 4187 Pst 1 4 246 1091 1666 3666 25 8010 21UM Chapter 7 Rev B 2006 24 Table 7 1 Quality control information for EGFP 2x FYVE cell line Table 7 2 Quality control information for the pCORON1000 EGFP 2x FYVE expression vector Table 7 3 Expected restriction pattern for the pCORON1000 EGFP 2x FYVE expression vector 8 Troubleshooting guide Problem 8 1 Low assay response positive vs negative controls 8 2 Low nuclear intensity 8 3 Image is out of focus 8 4 Cells do not adhere to well bottom in plate 8 5 Shading across image field 25 8010 21UM Chapter 8 Rev B 2006 Possible cause 8 1 1 Passage number too high 8 1 2 Cell density too low or too high 8 1 3 Incorrect selection of analysis parameters 8 1 4 Incorrect assay incubation conditions 8 1 5 Reagents were not stored properly or they are out of date 8 1 6 Cells have been stressed during assay 8 2 1 Nuclear stain concentration too low 8 2 2 Nuclear stain incubation time too short 8 3 1 Autofocus offset is chosen incorrectly or the system may need to be realigned 8 4 1 Plating density too high 8 5 1 Flatfield correction not applied or flatfield solution too weak 25 Remedy 8 1 1 Start a fresh bat
51. tions are performed at 37 C unless otherwise stated concentration of 100 nM is recommended However we recommend that users perform their own dose response curve to establish optimal agonist concentrations 3 Decant the Growth medium from the cell plate removing all excess liquid and add 200 ul Wash medium to wash the cells Decant the wash 4 Add 150 ul Assay medium 5 Add 50 ul of the prepared four fold dilution stocks of the test and control compounds to the appropriate wells The total well volume is 200 ul 6 After the first well has been incubated for 30 minutes read the assay plate using the IN Cell Analyzer 7 Perform the data analysis using the Granularity Analysis Module 5 3 Results 5 3 1 Calculating the Z factor Assay performance can be assessed by calculating the Z factor a dimensionless value defined by Zhang et al 19 Using the IN Cell Analyzer 3000 a Z factor of gt 0 6 should be obtained with the assay under standard conditions if the experiment is performed as described in this manual Z 1 30c 30c uc uc where standard deviation u mean signal c positive control c negative control 5 3 2 Example results The following figures Fig 5 3 and Fig 5 4 are taken from a single experiment to give the user an overall view of the images and results that can be obtained with the EGFP 2x FVYE assay using the IN Cell Analyzer 3000 Fig 5 3 shows an image taken
52. tor of cytokine induced signal transduction 17 The FYVE domain containing protein Fgd1 the transforming gene product of the faciogenital displasia gene has putative GEF activity and plays a role in Cdc42 mediated signalling to the actin cytoskeleton 1 3 EGFP 2x FYVE assay An assay has been developed using Redistribution technology to quantify the intracellular localization and translocation of an EGFP 2x FYVE fusion protein in stably transfected mammalian cell lines The EGFP 2X FYVE fusion protein used in this assay consists of the FYVE finger from the human homologue of the hepatocyte growth factor regulated tyrosine kinase substrate Hrs duplicated in tandem The 2x FYVE Redistribution assay monitors redistribution of EGFP 2x FYVE from its initial location bound to PI 3 P in early endosomes to the cytoplasm in cells challenged with test compounds This assay is optimized for image acquisition and analysis on the IN Cell Analyzer 3000 and IN Cell Analyzer 1000 using the Granularity Analysis Module although the assay can also be imaged on other systems The Granularity Analysis Module measures the degree of EGFP 2x FYVE localization on early endosomes by identifying granular fluorescence defined as focal regions within the cell having a defined intensity difference from their background On the addition of PI3K inhibitors such as Wortmannin which prevent the synthesis of PI 3 P EGFP 2x FYVE redistributes to the cytoplasm Fig
53. zen in the vapor phase of liquid Nitrogen 1 Remove a cryo vial from storage 2 Holding the cryo vial dip the bottom three quarters of the cryo vial into a 37 C water bath and swirl gently for 1 2 minutes until the contents are thawed Do not thaw the cells for longer than 3 minutes as this decreases viability 3 Remove the cryo vial from the water bath and wipe it with 70 v v Ethanol Transfer the cells immediately to a T 25 flask and add 5 ml pre warmed Growth medium drop wise to prevent cell damage Add a further 2 ml Growth medium and incubate at 37 C NOTE To ensure maximum cell viability do not allow the cells to thaw at room temperature and do not thaw the vial using your hands 5 1 4 Cell sub culturing procedure Incubation 5 CO 95 humidity 37 C The cells should be passaged in a ratio of 1 6 when they are 90 confluent 1 Warm all reagents to 37 C 2 Aspirate the medium from the cells and discard 3 Wash the cells with PBS Take care not to damage the cell layer while washing but ensure that the entire cell surface is washed 4 Aspirate the PBS from the cells and discard 5 Add Trypsin EDTA 2 ml for T 75 flasks and 4 ml for T 162 flasks ensuring that all cells are in contact with the solution Wait for 3 10 minutes for the cells to round up loosen Check on an inverted microscope 6 When the cells are loose tap the flask gently to dislodge the cells Add Growth medium 10 ml for T 75 and 8 m

EGFP-2x FYVE Assay - GE Healthcare Life Sciences

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