CytoSelect™ 96-Well Cell Transformation Assay
Contents
1. Product Manual CytoSelect 96 Well Cell Transformation Assay Soft Agar Colony Formation Catalog Number CBA 130 96 assays CBA 130 5 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Neoplastic transformation occurs via a series of genetic and epigenetic alterations that yield a cell population that is capable of proliferating independently of both external and internal signals that normally restrain growth For example transformed cells show reduced requirements for extracellular growth promoting factors are not restricted by cell cell contact and are often immortal Anchorage independent growth is one of the hallmarks of transformation which is considered the most accurate and stringent in vitro assay for detecting malignant transformation of cells Traditionally the soft agar colony formation assay is a common method to monitor anchorage independent growth which measures proliferation in a semisolid culture media after 3 4 weeks by manual counting of colonies Standard soft agar assays are usually performed in 100 mm or 60 mm dishes where cells are allowed to grow inside a semisolid culture media for 3 4 weeks before sizable colonies appear This method is quite cumbersome time consuming and difficult when testing a large number of samples Additionally the manual counting of colonies is highly subjective with varying2. at room temperature for 15 minutes Transfer 10 uL of the mixture to a 96 well plate suitable for fluorescence measurement 5 Add 90 uL of the CyQuant Working Solution to each well Incubate 10 minutes at room temperature Read the plate in a 96 well fluorometer using a 485 520 nm filter set eo CELL BIOLABS INC Example of Results The following figures demonstrate typical results with the CytoSelect 96 well Cell Transformation Assay Kit Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer Molecular Devices with a 485 538 nm filter set and 530 nm cutoff One should use the data below for reference only This data should not be used to interpret actual results 50 50 40 40 gt 30 gt 30 LL LL 20 20 10 10 0 0 0 1000 2000 3000 4000 0 5000 10000 15000 20000 25000 Cells mL x1000 Cells Figure 1 HeLa Cell Dose Curve Cervical carcinoma HeLa cells were resuspended at 4 x 10 cells mL and titrated 1 2 in culture medium followed by addition of Agar Solubilization Solution Lysis Buffer and Cyquant GR Dye detection as described in the Cell Dose Section Results were shown by cell concentration or by actual cell number in CyQuant Detection 20 7 HeLa ENIH3T3 15 gt L a 10 i 5 20000 10000 5000 2500 1250 625 313 0 Cells Seeded Well Figure 2 An
3. Human Breast Cancer Cells Carcinogenesis 10 1093 carcin bgs244 Maag D et al 2011 Inositol Polyphosphate Multikinase is a Physiologic PI3 Kinase that Activates Akt PKB PNAS 108 1391 1396 Inami Y et al 2011 Persistent Activation of Nrf2 through p62 in Hepatocellular Carcinoma Cells J Cell Biol 10 1083 jcb 201102031 CELL BIOLABS INC ae o gt 4 Rubio R et al 2010 Deficiency in p53 but not retinoblastoma induces the transformation of mesynchymal stem cells in vitro and initiates leiomyosarcoma in vivo Cancer Res 70 4185 4194 5 Faoro L et al 2010 EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival Cell Invasion Focal Adhesions and Mammalian Target of Rapamycin Activation J Biol Chem 285 18575 18585 6 Iorns E et al 2010 The Role of SATB1 in Breast Cancer Pathogenesis J Natl Cancer Inst 10 1093 jnci djq243 7 Liu F et al 2010 Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate Oncogene 29 3650 3664 8 Carnahan J 2010 Selective and Potent Raf Inhibitors Paradoxically Stimulate Normal Cell Proliferation and Tumor Growth Mol Cancer Ther 9 2399 2410 9 Kang M I et al 2009 A selective small molecule nuclear factor kB inhibitor from a high throughput cell based assay for activator protein 1 hits Mol Cancer Ther 8 571 581 10 Takezawa K et al 2009 Sorafenib inhibits non sm
4. all cell lung cancer cell growth by targeting B RAF in KRAS wild type cells and C RAF in RKAS mutant cells Cancer Res 69 6515 6521 11 Lee K B et al 2008 Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases Exp and Mol Medicine 40 1 118 129 12 Shen L et al 2008 E1A inhibits the proliferation of human cervical cancer cells HeLa cells by apoptosis induction through activation of HER 2 Neu Caspase 3 pathway Med Oncol 25 222 228 13 Wei Q et al 2008 Sulfiredoxin is an AP 1 target gene that is required for transformation and shows elevated expression in human skin malignancies PNAS 105 19738 19743 14 Gazin C et al 2007 An elaborate pathway required for Ras mediated epigenetic silencing Nature 449 7165 1073 1077 License Information CyQuant GR Dye is licensed from Molecular Probes Invitrogen Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any prox
5. chorage Independent Growth of HeLa Cells HeLa cells were seeded at various concentrations and cultured for 6 days HeLa cell transformation is determined according to the assay protocol CELL BIOLABS INC A A oies e r z e Fie Pra eo gt r A Ld s e P gt 6 r gt Ld S gt s Figure 3 HeLa Colony Formation HeLa cells were cultured for 14 days according to the assay protocol Colonies were visualized by 0 1 p iodonitro tetrazolium violet INT staining Calculation of Anchorage Independent Growth 1 Compare RFU values with the Cell Dose Curve and extrapolate the cell concentration in soft agar 2 Calculate the Total Transformed Cell Number Well Total Transformed Cells Well cells mL in soft agar x 0 125 mL well For example If you extrapolate your RFU value from your cell dose curve and determine you have 500 000 cells mL in your soft agar sample Total Transformed Cells Well 500 000 cells mL x 0 125 mL well 62 500 cells well References 1 Shin SI Freedman VH Risser R and Pollack R 1975 Proc Natl Acad Sci U S A 72 4435 9 2 Hahn WC Counter CM Lundberg AS Beijersbergen RL Brooks MW and Weinberg RA 1999 Nature 400 464 8 Recent Product Citations 1 Maccario C et al 2012 The Resveratrol Analog 4 4 Dihydroxy Trans Stilbene Suppresses Transformation in Normal Mouse Fibroblasts and Inhibits Proliferation and Invasion of
6. colony sizes it s difficult to determine meaningful results Cell Biolabs CytoSelect 96 well Cell Transformation Assay does not involve subjective manual counting of colonies or require a 3 4 week incubation period Instead cells are incubated only 6 8 days in a semisolid agar media before being solubilized lysed and detected by the patented CyQuant GR Dye in a fluorescence plate reader see Assay Principle below This format provides a quantitative high throughput method to accurately measure cell transformation Additionally the short incubation time 6 8 days makes it possible to assay cells transiently transfected with oncogenes or siRNA The CytoSelect 96 well Cell Transformation Kit provides a robust system for screening oncogenes and cell transformation inhibitors Each kit provides sufficient quantities to perform 96 tests in a microtiter plate CELL BIOLABS INC oa Assay Principle E Base Agar Layer A EE cen Agar Layer Base a rh ial faa IS e o Transformed Cells d c O Cell Colonies Agar Solubilization Agar Gelation Solution Fluorometric Detection Es Cell Agar Layer is Added lt gt Incubate 6 8 Days Formation of Cell Colonies Sa Addition of Agar Solubilization Solution lt J Homogeneous Cell Suspension Cells are lysed and detected with CyQuant GR dye J Fluorometric Detection Related Products CBA 106 CytoSelect 96 Well Cell Migration Assay 8um Fluorome
7. es to allow the cell agar layer to solidify III Quantitation of Anchorage Independent Growth 1 2 Add 100 uL of culture medium containing cell growth activator s or inhibitor s to each well Incubate the cells for 6 8 days at 37 C and 5 CO2 Examine the cell colony formation under a light microscope o gt CELL BIOLABS INC Remove culture medium by inverting the plate and blotting on paper towel Gently tap several times Add 50 uL of Agar Solubilization Solution to each well of the 96 well plate Incubate for 1 hr at 37 C Pipette each well 5 10 times to ensure complete agar solubilization Add 25 uL of 8X Lysis Buffer to each well Pipette each well 5 10 times to ensure a homogeneous mixture Incubate the plate at room temperature for 15 minutes 8 Transfer 10 uL of the mixture to a 96 well plate suitable for fluorescence measurement Add 90 uL of the CyQuant Working Solution to each well Incubate 10 minutes at room temperature 10 Read the plate in a 96 well fluorometer using a 485 520 nm filter set Cell Dose Curve optional 1 2 3 Harvest and resuspend cells in culture medium at 5 x 10 cells mL Prepare a serial of 2 fold dilution with culture medium including a medium blank Transfer 125 uL of each cell dilution to a microfuge tube Add 50 uL of Agar Solubilization Solution and 25 uL of 8X Lysis Buffer to each tube Vortex each tube to ensure a homogeneous mixture Incubate the tubes
8. imate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com CELL BIOLABS INC 2004 2012 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 10 N CELL BIOLABS INC JAN a
9. o the well e To avoid fast and uneven evaporation that leads to aberrant results we suggest not using the wells on the plate edge or filling the edge wells with medium to reduce evaporation Transfer the plate to 4 C for 30 minutes to allow the base agar layer to solidify 5 Prior to adding the Cell Agar Layer Section II allow the plate to warm up for 15 minutes at 37 C II Preparation of Cell Agar Layer samples should be assayed in triplicate 1 2 Melt 1 2 Agar Solution in a microwave and cool to 37 C in a water bath Warm 2X DMEM 20 FBS medium to 37 C in a water bath Allow at least 30 minutes for the temperature to equilibrate Harvest and resuspend cells in culture medium at 0 4 4 x 10 cells mL keep the cell suspension warm in a 37 C water bath Mix equal volumes of 1 2 Agar Solution 2X DMEM 20 FBS media and cell suspension 1 1 1 in a sterile pre warmed tube by inverting several times Immediately transfer 75 uL of the mixture to each well of the 96 well flat bottom microplate already containing the solidified base agar layer 25 uL of cell suspension containing 1000 10000 cells well will be seeded Note Work quickly with the agar solution to avoid gelation but gently pipette as not to disrupt the base layer integrity Also try to avoid adding air bubbles to the well Always include negative control wells that contain no cells in the cell agar layer Transfer the plate to 4 C for 15 minut
10. tion Place 1 2 g of Agar Powder in a sterile bottle add 100 mL of sterile cell culture grade water Microwave or boil until agar is completely dissolved 2X DMEM 20 FBS Medium In a sterile tube dilute the provided 5X DMEM in sterile cell culture grade water to 2X containing 20 FBS For example to prepare a 5 mL solution add 2 mL of 5X DMEM 1 mL of FBS and 2 mL of sterile cell culture grade water Sterile filter the 2X media to 0 2 um CyQuant Working Solution Immediately before use prepare sufficient amount of the CyQuant Working Solution by diluting the CyQuant GR Dye 1 400 with 1X PBS For example add 10 uL to 4 mL of 1X PBS Use the solution immediately do not store the CyQuant Working Solution CELL BIOLABS INC yA o gt Assay Protocol must be under sterile conditions I Preparation of Base Agar Layer 1 2 Melt 1 2 Agar Solution in a microwave and cool to 37 C in a water bath Warm 2X DMEM 20 FBS medium to 37 C in a water bath Allow at least 30 minutes for the temperature to equilibrate Mix equal volumes of 1 2 Agar Solution and 2X DMEM 20 FBS medium in a sterile pre warmed tube by inverting several times Immediately transfer 50 uL of the mixture to each well of a 96 well sterile flat bottom microplate Gently tap the plate a few times to allow the agar solution to evenly cover the wells Notes e Work quickly with the agar solution to avoid gelation Also try to avoid adding air bubbles t
11. tric CBA 106 C CytoSelect 96 Well Cell Migration and Invasion Assay 8um Fluorometric CBA 110 CytoSelect 24 Well Cell Invasion Assay Basement Membrane Colorimetric CBA 112 CytoSelect 96 Well Cell Invasion Assay Basement Membrane Fluorometric CBA 135 CytoSelect 96 Well Cell Transformation Assay Cell Recovery Colorimetric CBA 140 CytoSelect 96 Well Cell Transformation Assay Cell Recovery Fluorometric CBA 145 CytoSelect 384 Well Cell Transformation Assay CBA 150 CytoSelect In Vitro Tumor Sensitivity Assay CBA 155 CytoSelect Clonogenic Tumor Cell Isolation Kit 10 CBA 320 CytoSelect 96 Well Hematopoietic Colony Forming Cell Assay 3 CELL BIOLABS INC CO NDA RP wWN S Kit Components 1 CytoSelect Agar Powder Part No 113001 One bottle 1 2 g 2 5X DMEM Solution Part No 113002 Two sterile tubes 1 5 mL each 3 Agar Solubilization Solution Part No 113003 One amber bottle 6 0 mL 4 5 CyQuant GR Dye Part No 10103 One tube 25 uL 8X Lysis Buffer Part No 113004 One bottle 3 0 mL Materials Not Supplied Qo ee Ye YS Cells and Culture Medium 1X PBS 37 C Incubator 5 CO2 Atmosphere Light Microscope 96 well Fluorometer Microwave or Heating Block Water bath Optional Positive Control cells such as NIH 3T3 Ras G12V Storage Store all components at 4 C until their expiration dates Preparation of Reagents 1 2 Agar Solu
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