CytoSelect™ 96-Well In Vitro Tumor Sensitivity
Contents
1. Product Manual CytoSelect 96 Well In Vitro Tumor Sensitivity Assay Soft Agar Colony Formation Trial Size Catalog Number CBA 150 T 24 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Tumor sensitivity assays are intended to help predict the sensitivity of various tumors to chemotherapeutic agents with the intent of identifying the most effective treatment with the fewest side effects With this information physicians can devise tailor made chemotherapy regiments and eliminate ineffective drugs sparing patients of unnecessary toxicity Ideally an in vitro tumor sensitivity assay must be reliable sensitive and resemble the 3 D in vivo environment such as culturing in collagen gel or soft agar 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 Cell Biolabs CytoSelect 96 well In Vitro Tumor Sensitivity 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 solubilized transferred and detected by the provided MTT Solution in a microtiter plate reader see Assay Principle below The CytoSelect 96 well In Vitro Tumor S2. K et al 2012 HER2 amplification a potential mechanism of acquired resistance to EGFR inhibition in EGFR mutant lung cancers that lack the second site EGFRT790M mutation Cancer Discovery 2 922 933 Li C et al 2012 The root bark of Paeonia moutan is a potential anticancer agent in human oral squamous cell carcinoma cells Anticancer Res 32 2625 2630 Itamochi H et al 2011 Inhibiting the mTOR pathway synergistically enhances cytotoxicity in ovarian cancer cells induced by etoposide through upregulation of c Jun Clin Cancer Res 17 4742 4750 Kang D W et al 2010 Phospholipase D1 drives a positive feedback loop to reinforce the Wnt B catenin TCF signaling axis Cancer Res 70 4233 4242 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 65
3. in a water bath for 30 minutes or until agar matrix liquefies Transfer the tube 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 5 10 x 10 cells mL Prepare a serial 2 fold dilution in culture medium including a blank without cells following sequence In a sterile tube add the appropriate volume of 2X DMEM 20 FBS medium Transfer 50 uL of each dilution to a 96 well plate According to Table 3 below prepare the desired volume of Cell Dose Curve Solution in the a 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 Sterile Water CytoSelect 10X Total Volume of DMEM 20 mL Matrix Diluent CytoSelect Cell Dose Curve FBS Medium mL Agar Matrix Solution mL mL Solution mL 1 25 0 45 0 55 0 25 2 5 0 625 0 225 0 275 0 125 1 25 Table 3 Preparation of Cell Dose Curv
4. 00 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2013 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
5. HeLa Cell Dose Curve Cervical carcinoma HeLa cells were resuspended at 6 x 10 cells mL and titrated 1 2 in culture medium followed by addition of Cell Dose Curve Solution Matrix Solubilization Solution MTT Solution and Detergent Solution as described in the Cell Dose Section Results are shown by cell concentration or by actual cell number in MTT Detection 0 60 OD 570nm So S 0 20 0 10 0 00 11 9 7 5 3 Log 5 FU M Figure 2 Inhibition of Hela Cell Transformation by 5 Fluorouracil HeLa cells were seeded at 5000 cells well and cultured 7 days at various 5 FU concentrations Cell transformation was determined according to the assay protocol IC50 value of 5 Fluorouracil on HeLa cell anchorage independent growth was determined to be 1 uM 9 JaN CELL BIOLABS INC Ps s r z Figure 3 Inhibition of HeLa Cell Anchorage Independent Growth by Taxol HeLa cells were cultured for 7 days in the absence left or presence right of 1 nM Taxol according to the assay protocol Calculation of Anchorage Independent Growth 1 Compare ODs70nm 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 0 050 mL well For example If you extrapolate your ODs570nm value from your cell dose curve and determine you have 500 000 cells mL in your sample Total Transformed Cell
6. LL BIOLABS INC A Ss A 4 According to Table 2 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 DMEM 20 FBS Medium mL CytoSelect Matrix Diluent mL 10X CytoSelect Agar Matrix Solution mL Cell Suspension mL Total Volume of Cell Suspension Agar Matrix Layer mL of Tests in 96 well Plate 75 uL test 0 875 0 688 0 188 0 125 1 875 25 Table 2 Preparation of Cell Suspension Agar Matrix Layer After mixing incubate the Cell Suspension Agar Matrix Layer at room temperature for 5 minutes Immediately dispense 75 uL of Cell Suspension Agar Matrix Layer into each well of the 96 well 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
7. Solution tube to 90 95 C in a water bath for 30 minutes or until agar matrix liquefies Transfer the tube 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 agar matrix liquefies Transfer the tube 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 JA O CELL BIOLABS INC 3 According to Table 1 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 Solution is slightly viscous care should be taken in accurately pipetting the appropriate volume 2X DMEM 20 Sterile Water 10X Total Volume of of Tests in 96 FBS Medium mL CytoSelect Base Agar Matrix well Plate 50 mL Agar Matrix Layer mL uL test Solution mL 0 625 0 5 0 125 1 25 25 Table 1 Preparation of Base Agar Matrix Layer Af
8. e Solution 7 N JA ZA CELL BIOLABS INC 10 11 12 13 14 15 Immediately dispense 125 uL of Cell Dose Curve Solution into the wells of the 96 well plate already containing the cell serial dilution from step 5 Add 125 uL of 1X Matrix Solubilization Buffer to each well Pipette each well 10 12 times to mix thoroughly Transfer 100 uL of the mixture to a 96 well microtiter plate Add 10 uL of MTT Solution to each well Pipette each well 7 10 times to ensure a homogeneous mixture Incubate the plate for 2 4 hours at 37 C and 5 CO2 Note Under the microscope a purple precipitate should be visible within the cells Add 100 uL of Detergent Solution to each well Incubate the plate in the dark for 2 4 hours at room temperature Pipette each well 7 10 times to ensure a homogeneous mixture Measure the absorbance at 570 nm in a 96 well microtiter plate reader Example of Results The following figures demonstrate typical results with the CytoSelect Cell Transformation Assay Kit Absorbance measurements were performed on a Microplate Autoreader EL311 Bio Tek Instruments Inc with a 570 nm filter One should use the data below for reference only This data should not be used to interpret actual results CELL BIOLABS INC 1 6 1 2 c 0 8 1O m 00 4 r r 0 2000 4000 6000 0 2000 4000 6000 Cells mL x 1000 Cells mL x 1000 Figure 1
9. elect Clonogenic Tumor Cell Isolation Kit 10 CBA 320 CytoSelect 96 Well Hematopoietic Colony Forming Cell Assay 3 oe BIOLABS INC Kit Components SS Ee ey SS 10X CytoSelect Agar Matrix Solution Part No 114001 T One 1 mL sterile tube CytoSelect Matrix Diluent Part No 114002 T One 1 mL sterile tube 5X DMEM Solution Part No 113002 One 1 5 mL sterile tube 1X Matrix Solubilization Buffer Part No 115001 T Three 2 mL sterile tubes Detergent Solution Part No 113501 T Two 1 5 mL tubes MTT Solution Part No 113502 T One 0 3 mL tube Materials Not Supplied 1 Tumor Cells cancer cell line or cells prepared from solid tumor 2 Anticancer Agents e g Taxol 5 Fluorouracil anticancer mAb or siRNA 3 37 C Incubator 5 CO2 Atmosphere 4 Light Microscope 5 96 well Microtiter Plate Reader 6 37 C and boiling water baths Storage Store all components at 4 C until their expiration dates Preparation of Reagents 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 1 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 10X CytoSelect Agar Matrix Solution Heat the Agar Matrix
10. ensitivity Assay provides a stringent anchorage independent model for chemosensitivity testing and potential anticancer drug screening Each Trial Size kit provides sufficient quantities to perform 24 tests in a 96 well microtiter plate 2 CELL BIOLABS INC k Assay Principle LO Base Agar Matrix Layer E Cell Suspension Agar Matrix Layer Transformed Cells O Cell Colonies Matrix Solubilization Solution BB Colorimetric Detection Related Products oe Pe a eee Base Agar Matrix is Added Agar Gelation Cell Suspension Agar Matrix is Added wW Incubate 6 8 Days Formation of Cell Colonies Jat Addition of Matrix Solubilization Solution ccs fel Cells are transfered and detected with MTT Solution l J Colorimetric Detection CBA 100 CytoSelect 24 Well Cell Migration Assay 8um Colorimetric CBA 106 CytoSelect 96 Well Cell Migration Assay 8um Fluorometric 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 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 155 CytoS
11. s Well 500 000 cells mL x 0 050 mL well 25 000 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 2 3 Joshi P et al 2015 MicroRNA 148a reduces tumorigenesis and increases TRAIL induced apoptosis in NSCLC Proc Natl Acad Sci U S A 112 8650 8655 Meador C B et al 2015 Optimizing the sequence of anti EGFR targeted therapy in EGFR mutant lung cancer Mol Cancer Ther 14 542 552 Peng Y T et al 2015 Upregulation of cyclin dependent kinase inhibitors CDKNIB and CDKNIC in hepatocellular carcinoma derived cells via goniothalamin mediated protein stabilization and epigenetic modifications Toxicol Rep doi 10 1016 j toxrep 2015 01 010 Akl M R et al 2015 Araguspongine C induces autophagic death in breast cancer cells through suppression of c Met and HER2 receptor tyrosine kinase signaling Mar Drugs 13 288 311 10 JA CELL BIOLABS INC A aise SN 10 Suman S et al 2014 Activation of AKT signaling promotes epithelial mesenchymal transition and tumor growth in colorectal cancer cells Mol Carcinog 53 E151 E160 Bard Chapeau E et al 2013 EVIL oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation PNAS 110 E2885 E2894 Takezawa
12. ter mixing maintain the Base Agar Matrix Layer at 37 C to avoid gelation 5 Dispense 50 uL of Base Agar Matrix Layer into each well of a 96 well sterile flat bottom microplate samples should be assayed in triplicate Gently tap the plate a few times to ensure the Base Agar Matrix Layer evenly covers the wells Notes e Work quickly with the layer 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 6 Transfer the plate to 4 C for 30 minutes to allow the Base Agar Matrix Layer to solidify 7 Prior to adding the Cell Suspension Agar Matrix Layer Section ID allow the plate to warm to room temperature for 30 minutes II Addition of Cell Suspension Agar Matrix Layer under sterile conditions 1 2 Heat the 10X CytoSelect Agar Matrix Solution to 90 95 C in a water bath for 30 minutes or until agar matrix liquefies Transfer the tube 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 1 1 x 10 cells mL Keep the cell suspension warm in a 37 C water bath 5 CE
13. well e Always include negative control wells that contain no cells in the Cell Suspension Agar Matrix Layer Transfer the plate to 4 C for 20 minutes to allow the Cell Suspension Agar Matrix Layer to solidify 8 Allow the plate to warm to room temperature for 30 minutes 10 Add 50 uL of culture medium containing anticancer agents e g Taxol 5 Fluorouracil mAb etc to each well Incubate the cells for 6 8 days at 37 C and 5 CO2 Examine the colony formation under a light microscope III Quantitation of Anchorage Independent Growth 1 2 Add 125 uL of the 1X Matrix Solubilization Buffer to each well Pipette the entire volume of the well 10 12 times to mix thoroughly and solubilize the agar matrix completely 6 CELL BIOLABS INC A a 3 Transfer 100 uL of the mixture to a 96 well microtiter plate oS a O Add 10 uL of MTT Solution to each well Pipette each well 7 10 times to ensure a homogeneous mixture Incubate the plate for 2 4 hours at 37 C and 5 CO3 Note Under the microscope a purple precipitate should be visible within the cells Add 100 uL of Detergent Solution to each well Incubate the plate in the dark for 2 4 hours at room temperature Pipette each well 7 10 times to ensure a homogeneous mixture Measure the absorbance at 570 nm in a 96 well microtiter plate reader Cell Dose Curve optional 1 2 3 4 Heat the 10X CytoSelect Agar Matrix Solution to 90 95 C
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