CytoSelect™ 96-Well Cell Transformation Assay
Contents
1. 20000 10000 5000 2500 1250 625 313 0 Cells Seeded Well Figure 2 Anchorage 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 we sae gt e yee gt i AN ee a gt F e 19 r gt es lt 6 gt Ea e gt gt 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 2 Shin SI Freedman VH Risser R and Pollack R 1975 Proc Natl Acad Sci U S A 72 4435 9 Hahn WC Counter CM Lundberg AS Beijersbergen RL Brooks MW and Weinberg RA 1999 Nature 400 464 8 Recent Product Citations 1 2 3 Ercan D et al 2015 EGFR mutations and resistance to irreversible p2. Product Manual CytoSelect 96 Well Cell Transformation Assay Soft Agar Colony Formation Trial Size Catalog Number CBA 130 T 24 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 varying colony size
3. 1 2 Agar Solution Place 0 24 g of Agar Powder in a sterile bottle then add 20 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 2 5 mL solution add 1 mL of 5X DMEM 0 5 mL of FBS and mL of sterile cell culture grade water Sterile filter the 2X media to 0 2 um Note You may substitute your own medium in place of the DMEM we provide but ensure that it is at a 2X concentration 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 4 les CELL BIOLABS INC A a 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
4. cells Potential drug synergism with gemcitabine Mol Med Rep doi 10 3892 mmr 2015 3589 He C et al 2015 YAP forms autocrine loops with the ERBB pathway to regulate ovarian cancer initiation and progression Oncogene doi 10 1038 onc 2015 52 Kim T et al 2015 Role of MYC regulated long noncoding RNAs in cell cycle regulation and tumorigenesis J Natl Cancer Inst 107 dju505 Galvan A et al 2015 Germline polymorphisms and survival of lung adenocarcinoma patients a genome wide study in two european patient series Int J Cancer 136 E262 E271 Nakanishi Y et al 2015 Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3 BAIAP2L1 Mol Cancer Ther doi 10 1158 1535 7163 MCT 14 0927 T Westbom C M et al 2014 CREB induced inflammation is important for malignant mesothelioma growth Am J Pathol 184 2816 2827 Kong L Y et al 2014 Therapeutic targets in subependymoma J Neuroimmunol 277 168 175 Takiguchi S et al 2014 Involvement of CXCL14 in osteolytic bone metastasis from lung cancer Int J Oncol 44 1316 1324 Wang L et al 2014 Ectopic over expression of miR 429 induces mesenchymal to epithelial transition MET and increased drug sensitivity in metastasizing ovarian cancer cells Gynecol Oncol 134 96 103 Sun Y et al 2014 Cholesterol induced activation of TRPM7 regulates cell proliferation migration and viability of human prostate cells Biochim Biophys Acta 1843 1839 1
5. the well Always include negative control wells that contain no cells in the cell agar layer Transfer the plate to 4 C for 15 minutes 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 CO Examine the cell colony formation under a light microscope 5 CELL BIOLABS INC A a 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 5 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 1 5 x 10 cells mL Prepare a 2 fold serial dilution with culture medium including a medium blank Transfer 125 uL of each cell dilution to a microfuge tube Add 50 uL
6. 3 carcin bgs244 License Information This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased product and components of the product only in research conducted by the buyer whether the buyer is an academic or for profit entity The sale of this product is expressly conditioned on the buyer not using the product or its components or any materials made using the product or its components in any activity to generate revenue which may include but is not limited to use of the product or its components i in manufacturing 11 to provide a service information or data in return for payment Gii for therapeutic diagnostic or prophylactic purposes or iv for resale regardless of whether they are resold for use in research For information on purchasing a license to this product for purposes other than as described above contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com 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 purchas
7. 850 Priya S et al 2014 Diethyl maleate inhibits MCA TPA transformed cell growth via modulation of GSH MAPK and cancer pathways Chem Biol Interact 219 37 47 Nakamura T et al 2014 Osteopontin integrin av 3 axis is crucial for 5 fluorouracil resistance in oral squamous cell carcinoma FEBS Lett 589 231 239 Eckerdt F et al 2014 Regulatory effects of a Mnk2 eIF4E feedback loop during mTORC1 targeting of human medulloblastoma cells Oncotarget 5 8442 8451 He X et al 2014 Involvement of polypyrimidine tract binding protein PTBP1 in maintaining breast cancer cell growth and malignant properties Oncogenesis 3 e84 Lin L et al 2014 A large scale RNAi based mouse tumorigenesis screen identifies new lung cancer tumor suppressors that repress FGFR signaling Cancer Discov 4 1168 1181 Zecchini V et al 2014 Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer EMBO J 33 1365 1382 Eisfeld A K et al 2014 NRAS isoforms differentially affect downstream pathways cell growth and cell transformation PNAS 111 4179 4184 Gong J et al 2014 Combined targeting of STAT3 NF kB COX 2 EP4 for effective management of pancreatic cancer Clin Cancer Res 20 1259 1273 Kegelman T et al 2014 MDA 9 Syntenin is a key regulator of glioma pathogenesis Neuro Oncology 16 50 61 9 JaN CELL BIOLABS INC AO _ 28 Giacoia E G et al Targeting Plasminogen Acti
8. Select 96 Well Cell Migration Assay 8um Fluorometric 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 Kit Components 1 CytoSelect Agar Powder Part No 113001 T One 0 24 g tube 5X DMEM Solution Part No 113002 One 1 5 mL sterile tube 2 3 Agar Solubilization Solution Part No 113003 T One 1 5 mL amber tube 4 5 CyQuant GR Dye Part No 10103 T One 10 uL tube 8X Lysis Buffer Part No 113004 T One 1 mL tube Materials Not Supplied Qo ee YS 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
9. er 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 proximate incidental or consequential damages in connection with the products 10 J _ CELL BIOLABS INC n 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 2014 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 11 CELL BIOLABS INC Wa SN
10. 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 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 6 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 40 30 20 10 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 1 HeLa NIH3T3 15 L am 10 7 l 5
11. s 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 Trial Size kit provides sufficient quantities to perform 24 tests in a 96 well microtiter plate CELL BIOLABS INC a Assay Principle E Base Agar Layer E Cell Agar Layer Transformed Cells O Cell Colonies Agar Solubilization Solution E Fluorometric Detection Cells are lysed and detected with CyQuant GR dye Related Products D o No oe e U N o Agar Gelation Incubate 6 8 Days Addition of Agar Solubilization Solution wW Jat ea L l y i Base Agar Layer is Added Cell Agar Layer is Added Formation of Cell Colonies Homogeneous Cell Suspension Fluorometric Detection CBA 106 Cyto
12. solution to evenly cover the wells Notes e Work quickly with the agar solution to avoid gelation Also try to avoid adding air bubbles to 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
13. vator Inhibitor 1 inhibits angiogenesis and tumor growth in a human cancer xenograft model Mol Cancer Ther 12 2697 2708 29 Hatano K et al 2013 Residual prostate cancer cells after docetaxel therapy increase the tumorigenic potential via constitutive signaling of CKCR4 ERK1 2 and c Myc Mol Cancer Res 11 1088 1100 30 Gupta P et al 2013 Oncrasin targets the JNK NF B axis to sensitize glioma cells to TNFa induced apoptosis Carcinogenesis 34 388 396 31 Xing C et al 2013 Reversing effect of ring finger protein 43 inhibition on malignant phenotypes of human hepatocellular carcinoma Mol Cancer Ther 12 94 103 32 Rai V et al 2012 Lysophosphatidic acid targets vascular and oncogenic pathways via RAGE signaling J Exp Med 209 2339 2350 33 Ghantous A et al 2012 Inhibition of tumor promotion by parthenolide epigenetic modulation of p21 Cancer Prevention Research 5 1298 1309 34 Shin S Y et al 2012 Transcriptional regulation of the interleukin 11 gene by oncogenic Ras Carcinogenesis 10 1093 carcin bgs297 35 Tan X et al 2012 cAMP response element binding protein promotes gliomagenesis by modulating the expression of oncogenic microRNA 23a PNAS 109 15805 15810 36 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 human breast cancer cells Carcinogenesis 10 109
14. yrimidine based EGFR inhibitors Clin Cancer Res 21 3913 3923 Mayr C et al 2015 3 Deazaneplanocin A may directly target putative cancer stem cells in biliary tract cancer Anticancer Res 35 4697 4705 He C et al 2015 The Hippo Y AP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression EMBO Mol Med doi 10 15252 emmm 201404976 Hua G et al 2015 YAP induces high grade serous carcinoma in fallopian tube secretory epithelial cells Oncogene doi 10 1038 onc 2015 288 Ukaji T et al 2015 Inhibition of IGF 1 mediated cellular migration and invasion by migracin A in ovarian clear cell carcinoma cells PLoS One 10 c0137663 8 AN 5 CELL BIOLABS INC JE a 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 2T Fujita T et al 2015 Identification and characterization of CXCR4 positive gastric cancer stem cells PLoS One 10 e0130808 Bon H et al 2015 Salt inducible Kinase 2 regulates mitotic progression and transcription in prostate cancer Mol Cancer Res 13 620 635 Yonesaka K et al 2015 Anti HER3 monoclonal antibody patritumab sensitizes refractory non small cell lung cancer to the epidermal growth factor receptor inhibitor erlotinib Oncogene doi 10 1038 onc 2015 142 Mayr C et al 2015 Cytotoxic effects of chemokine receptor 4 inhibition by AMD3100 in biliary tract cancer
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