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CytoSelect™ 384-Well Cell Transformation Assay

1. Product Manual CytoSelect 384 Well Cell Transformation Assay Fluorometric Catalog Number CBA 145 384 assays CBA 145 5 5 x 384 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
2. 5 After mixing incubate the Cell Suspension Agar Matrix Layer at room temperature for 5 minutes Immediately dispense 40 uL of Cell Suspension Agar Matrix Layer into each well of the tissue culture plate already containing the Base Agar Matrix Layer Section I Notes e Work quickly with the layer to avoid gelation but gently pipette as not to disrupt the base layer integrity Also try to avoid adding air bubbles to the well e Always include negative control wells that contain no cells in the Cell Suspension A gar Matrix Layer to solidify 8 Allow the plate to warm to room temperature for 30 minutes 10 Transfer the plate to 4 C for 10 minutes to allow the Cell Suspension Agar Matrix Layer Add 20 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 colony formation under a light microscope eee CELL BIOLABS INC III Quantitation of Anchorage Independent Growth 1 Prepare sufficient 5X Lysis Buffer CyQuant GR dye solution for all samples by diluting the dye 1 60 in 5X Lysis Buffer for example add 10 uL dye to 590 uL of 5X Lysis Buffer Add 20 uL of 5X Lysis Buffer CyQuant GR dye solution to each well Incubate the plate at room temperature for 30 minutes 3 Pipette each well 7 10 times to ensure a homogeneous mixture Read the plate in a 384 well fluorometer using a 485 520 nm filte
3. CBA 106 C CytoSelect 96 Well Cell Migration and Invasion Assay 8um Fluorometric CBA 112 CytoSelect 96 Well Cell Invasion Assay Basement Membrane Fluorometric CBA 130 CytoSelect 96 Well Cell Transformation Assay Soft Agar Colony Formation CBA 135 CytoSelect Cell Transformation Assay Cell Recovery Colorimetric CBA 140 CytoSelect 96 Well Cell Transformation Assay Cell Recovery Fluorometric CBA 150 CytoSelect In Vitro Tumor Sensitivity Assay 3 Sa oN ee Ye a CELL BIOLABS INC LOE SE 9 CBA 155 CytoSelect Clonogenic Tumor Cell Isolation Kit 10 CBA 320 CytoSelect 96 Well Hematopoietic Colony Forming Cell Assay Kit Components 1 2 3 4 5 CyQuant GR Dye Part No 10105 Two tubes 75 uL each 10X CytoSelect Agar Matrix Solution Part No 114001 One sterile bottle 10 0 mL CytoSelect Matrix Diluent Part No 114501 One sterile bottle 10 0 mL 5X DMEM Solution Part No 114502 One sterile bottle 10 0 mL 5X Lysis Buffer Part No 114503 One bottle 20 0 mL Materials Not Supplied et Oy oY a ee Cells and Culture Medium 37 C Incubator 5 CO Atmosphere Light Microscope Sterile 384 well Fluorometer Plate 384 well Fluorometer 37 C and boiling water baths Optional Positive Control cells such as NIH 3T3 Ras G12V Storage Store all components at 4 C until their expiration dates Preparation of Reagents 2X DMEM 2
4. is optional Transfer the bottle to a 37 C water bath for 20 minutes and maintain until needed Warm the 2X DMEM 20 FBS medium see Preparation of Reagents section and CytoSelect Matrix Diluent 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 5 5 x 10 cells mL Keep the cell suspension warm in a 37 C water bath CELL BIOLABS INC eee 4 According to Table 3 below prepare the desired volume of Cell Suspension Agar Matrix Layer in the following sequence In a sterile tube add the appropriate volume of 2X DMEM 20 FBS medium b Next add the corresponding volume of CytoSelect Matrix Diluent Mix well c Next add the corresponding volume of 10X CytoSelect Agar Matrix Solution Mix well d Finally add the corresponding volume of cell suspension Mix well Note The CytoSelect Matrix Diluent and 10X CytoSelect Agar Matrix Solution are slightly viscous care should be taken in accurately pipetting the appropriate volumes 2X CytoSelect 10X Cell Total Volume of of Tests in DMEM 20 Matrix Diluent CytoSelect Suspension Cell Suspension 384 well Plate FBS Medium mL Agar Matrix mL Agar Matrix 40 uL test mL Solution mL Layer mL 7 5 5 9 1 6 1 16 400 3 75 2 95 0 8 0 5 8 200 1 875 1 475 0 4 0 25 4 100 Table 3 Preparation of Cell Suspension Agar Matrix Layer
5. 0 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 10X CytoSelect Agar Matrix Solution Heat the Agar Matrix Solution bottle to 90 95 Cin a water bath for 30 minutes or until agarose liquefies microwaving is optional Transfer the bottle to a 37 C water bath for 20 minutes and maintain until needed Assay Protocol must be under sterile conditions I Preparation of Base Agar Matrix Layer 1 Heat the 10X CytoSelect Agar Matrix Solution to 90 95 C in a water bath for 30 minutes or until agarose liquefies microwaving is optional Transfer the bottle to a 37 C water bath for 20 minutes and maintain until needed 2 Warm the 2X DMEM 20 FBS medium see Preparation of Reagents section to 37 C in a water bath Allow at least 30 minutes for the temperature to equilibrate 4 gt CELL BIOLABS INC E oe 3 According to Table 2 below prepare the desired volume of Base Agar Matrix Layer in the following sequence In a sterile tube add the appropriate volume of 2X DMEM 20 FBS medium b Next add the corresponding volume of sterile water Mix well c Finally add the corresponding volume of 10X CytoSelect Agar Matrix Solution Mix well Note The 10X CytoSelect Agar Matrix
6. Solution mL Solution mL 1 0 0 315 0 485 0 2 2 0 0 5 0 158 0 242 0 1 1 0 Table 4 Preparation of Cell Dose Curve Solution CELL BIOLABS INC E oe eo 7 Immediately dispense 60 uL of Cell Dose Curve Solution into the wells of the 384 well plate already containing the cell serial dilution from step 5 8 Prepare sufficient 5X Lysis Buffer CyQuant GR dye solution for all samples by diluting the dye 1 60 in 5X Lysis Buffer for example add 10 uL dye to 590 uL of 5X Lysis Buffer 9 Add 20 uL of 5X Lysis Buffer CyQuant GR dye solution to each well Incubate the plate at room temperature for 30 minutes 10 Pipette each well 7 10 times to ensure a homogeneous mixture 11 Read the plate in a 384 well fluorometer using a 485 520 nm filter set Example of Results The following figures demonstrate typical results with the CytoSelect 384 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 RFU 600 600 500 500 400 400 gt 2000 4000 6000 300 i 300 200 cc 200 100 0 100 0 500 1000 1500 2000 2500 0 Cells mL x 1000 O 10000 ERARA 40000 50000 Figure 1 HeLa Cell Dose Curve Cervical carcinoma HeLa cells
7. Solution is slightly viscous care should be taken in accurately pipetting the appropriate volume 2X DMEM 20 Sterile Water 10X CytoSelect Total Volume of of Tests in FBS Medium mL Agar Matrix Base Agar 384 well Plate mL Solution mL Matrix Layer 20 uL test mL 4 3 2 0 8 8 400 2 1 6 0 4 4 200 1 0 8 0 2 2 100 Table 2 Preparation of Base Agar Matrix Layer After mixing maintain the Base Agar Matrix Layer at 37 C to avoid gelation 5 Dispense 20 uL of Base Agar Matrix Layer into each well of a sterile 384 well fluorometer plate samples should be assayed in triplicate Gently tap the plate a few times to ensure the Base Agar Matrix Layer evenly covers the wells Note 1 Work quickly with the layer to avoid gelation Also try to avoid adding air bubbles to the well 2 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 20 minutes to allow the Base Agar Matrix Layer to solidify 7 Prior to adding the Cell Suspension Agar Matrix Layer Section II allow the plate to warm to room temperature for 30 minutes II Addition of Cell Suspension Agar Matrix Layer under sterile conditions 1 Heat the 10X CytoSelect Agar Matrix Solution to 90 95 C in a water bath for 30 minutes or until agarose liquefies microwaving
8. n RL Brooks MW and Weinberg RA 1999 Nature 400 464 8 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 proximate 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 2006 2011 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 10 CELL BIOLABS INC r
9. r set Cell Dose Curve optional 1 a ee e a Heat the 10X CytoSelect Agar Matrix Solution to 90 95 C in a water bath for 30 minutes or until agarose liquefies microwaving is optional Transfer the bottle to a 37 C water bath for 20 minutes and maintain until needed Warm the 2X DMEM 20 FBS medium see Preparation of Reagents section and CytoSelect Matrix Diluent 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 1 5 x 10 cells mL Prepare a serial 2 fold dilution in culture medium including a blank without cells Transfer 20 uL of each dilution to a 384 well fluorometer plate According to Table 4 below prepare the desired volume of Cell Dose Curve Solution in the following sequence a Ina sterile tube add the appropriate volume of 2X DMEM 20 FBS medium b Next add the corresponding volume of sterile water Mix well c Next add the corresponding volume of CytoSelect Matrix Diluent Mix well d Finally add the corresponding volume of 10X CytoSelect Agar Matrix Solution Mix well Note The CytoSelect Matrix Diluent and 10X CytoSelect Agar Matrix Solution are slightly viscous care should be taken in accurately pipetting the appropriate volumes 2X DMEM 20 Sterile Water CytoSelect Matrix 10X CytoSelect Total Volume of FBS Medium mL Diluent mL Agar Matrix Cell Dose Curve mL
10. s it s difficult to determine meaningful results The CytoSelect 384 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 proprietary semisolid agar media before being 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 trans formation while the short incubation time makes it possible to assay cells transiently transfected with oncogenes or siRNA The CytoSelect 384 well Cell Transformation Assay provides a stringent anchorage independent model for measuring in vitro drug sensitivity and screening cell transformation inhibitors Each kit provides sufficient quantities to perform 384 tests in a 384 well plate CELL BIOLABS INC Assay Principle Base Agar Matrix is Added E Base Agar Matrix Layer H cei Suspension Agar Matrix Layer e Transformed Cells cell Colony Agar Gelation m CyQuant Detection Patent Pending Cell Suspension Agar Matrix is Added Incubate 6 8 days Formation of Cell Colonies Cells are lysed and detected with CyQuant GR Dye Fluorometric Detection Related Products CBA 100 CytoSelect 24 Well Cell Migration Assay 8um Colorimetric CBA 106 CytoSelect 96 Well Cell Migration Assay 8um Fluorometric
11. were resuspended at 2 5 x 10 cells mL and titrated 1 2 in culture medium followed by addition of Cell Dose Curve Solution Lysis Buffer and Cyquant GR Dye detection as described in the Cell Dose Section Results are shown by cell concentration or by actual cell number in CyQuant Detection CELL BIOLABS INC RFU 700 600 500 400 300 200 iv H i 0 313 156 78 0 1250 625 Cells Seeded Well Figure 2 Anchorage Independent Growth of HeLa Cells HeLa cells were seeded at various concentrations and cultured for 7 days Cell transformation was determined according to the assay protocol Figure 3 Colony Formation HeLa cells were cultured in semisolid agar media in a 96 well plate Photograph of cell colonies was taken after a 7 day culture Calculation of Anchorage Independent Growth 1 Compare RFU values with the Cell Dose Curve and extrapolate the cell concentration 2 Calculate the Total Transformed Cell Number Well Total Transformed Cells Well cells mL x 0 020 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 sample Total Transformed Cells Well 500 000 cells mL x 0 020 mL well 10 000 cells well 9 Jau BIOLABS INC 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 Beijersberge

CytoSelect™ 384-Well Cell Transformation Assay

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