CytoSelect™ Clonogenic Tumor Cell Isolation Kit
Contents
1. 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 150 CytoSelect 96 Well In Vitro Tumor Sensitivity Assay Soft Agar Colony Formation 10 CBA 320 CytoSelect 96 Well Hematopoietic Colony Forming Cell Assay Kit Components Ny ee oY SS 10X CytoSelect Agar Matrix Solution Part No 114001 One sterile bottle 10 0 mL CytoSelect Matrix Diluent Part No 114002 One sterile bottle 4 0 mL 5X DMEM Medium Part No 20103 One sterile bottle 5 0 mL 100X Osmotic Lysis Buffer Part No 115501 One sterile tube 1 0 mL 10X Assay Buffer Part No 115502 One sterile bottle 15 0 mL Filters Part No 115503 Five sterile filters Materials Not Supplied 1 Solid Tumor 2 Tumor Digestion Enzyme Mixture containing Collagenase DNase 3 Razor blade or scalpel 4 5 6 7 70 100 um sterile mesh filter or Cell Strainer 37 C Incubator 5 CO Atm2. Product Manual CytoSelect Clonogenic Tumor Cell Isolation Kit Catalog Number CBA 155 5 preps CBA 155 5 5 x 5 preps 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 Anchorage independence is an important hallmark of the transformation that correlates with tumorgenicity It has been shown that cells from most types of human solid tumors can form colonies in soft agar culture with plating efficiency from 0 01 to 1 furthermore cells recovered from individual colonies can form tumors when transplanted into mice Stem cells are defined as clonogenic cells capable of both self renewal and multilineage differentiation Recent data from both hematologic malignancies and solid tumors have suggested that there are only minor populations of cells in each malignancy that are capable of tumor initiation These tumor initiating cells have the functional properties of a tumor stem cell They appear to be capable of asymmetric division and self renewal and are only a minor faction among the bulk of more differentiated cells in the tumor These observations have profound implications for tumor biology research as well as successful t
3. 463 4 Al Hajj M Wicha MS Benito Hernandez A Morrison SJ Clarke MF Proc Natl Acad Sci 2003 100 7 3983 8 5 Reya T Morrison SJ Clarke MF Weissman IL Nature 2001 414 105 111 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 2007 2011 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 11 eo CELL BIOLABS INC J AU
4. Layer uL well Culture Media uL well 1000 Table 1 Dispensing Volumes I Solid Tumor Preparation 1 Dissect tumor and place into a 10 cm petri dish with 5 10 mL of desired media e g HBSS RPMI 2 Quickly mince the tumor into small fragments with a scalpel razor blade pieces should be approximately 1 3 mm 3 To further dissociate single cells from the tumor fragments add tumor digestion enzyme mixture to the media and mix well 4 Incubate the pieces at 37 C for 1 3 hours periodically mixing shaking the solution 5 Titurate the fragment mixture 3 5 times with a 10 mL pipette 6 Pass the solution through a 70 100 um sterile mesh filter not provided to remove any large undigested tumor pieces 7 Collect the filtered solution CELL BIOLABS INC P o gt 8 Pellet the cells by centrifugation at 1200 rpm for 5 minutes 10 11 12 13 14 Aspirate the supernatant Resuspend the pellet in 9 mL of 1X Osmotic Lysis Buffer for 30 seconds mixing well Note This step removes most of the contaminating RBC Immediately add 1 mL of 10X Assay Buffer mixing well Pellet the remaining cells at 1200 rpm for 5 minutes Aspirate the supernatant and resuspend the cells in 2 mL of complete culture medium Determine the cell concentration and adjust to 2 5 x 10 cells mL II Preparation of Base Agar Matrix Layer 1 Heat the 10X CytoSelect Agar Matrix So
5. ix 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 CytoSelect Suspension Cell Suspension 6 well Plate FBS Medium Diluent mL Agar Matrix mL Agar Matrix 1 5 mL test mL Solution mL Layer mL 3 5 2 75 0 75 0 5 7 5 5 2 1 1 65 0 45 0 3 4 5 3 0 7 0 55 0 15 0 1 1 5 1 Table 3 Preparation of Cell Suspension Agar Matrix Layer 5 After mixing incubate the Cell Suspension Agar Matrix Layer at room temperature for 5 minutes CELL BIOLABS INC Immediately dispense 1 5 mL of Cell Suspension Agar Matrix Layer into each well of the 6 well plate already containing the Base Agar Matrix Layer Section II 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 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 Carefully add 1 mL of culture medium to each well in a dropwise fashion as to not disturb the layers Incubate the cells for 6 8 days a
6. lter chamber sereval times Remove the inside media and transfer to a clean conical tube At this point colonies can either be directly transferred to a tissue culture flask dish or typsinized to create a single cell suspension before transferring eo CELL BIOLABS INC a Example of Results The following figures demonstrate typical results with the CytoSelect Clonogenic Tumor Cell Isolation Kit One should use the data below for reference only This data should not be used to interpret actual results Figure 1 Clonogenic Colony Formation Isolation and Re plating Mouse lung tumor was excised minced and digested before seeding 500 000 cells well in agar matrix suspension according to the assay protocol Panel A demonstrates clonogenic colony formation after 7 days red arrows show colonies black arrows show single cells Panel B demonstrates recovered clonogenic colonies single cells have been removed Panel C demonstrates re plated clonogenic colonies after 3 days without prior typsinization Panel D demonstrates re plated clonogenic colonies after 1 day colonies were typsinized titurated to create a single celled suspension before plating References 1 Pavelic ZP Slocum HK Rustum YM Creaven PJ Nowak NJ Karakousis C Takita H Mittelman A Cancer Res 1980 40 11 4151 8 2 Heppner G H Cancer Res 1984 44 2259 2265 10 Jau BIOLABS INC Hamburger A W Salmon S E Science 1977 197 461
7. lution 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 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 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 FBS Medium mL CytoSelect Base Agar Matrix 6 well Plate mL Agar Matrix Layer mL 1 mL test Solution mL 2 5 2 0 5 5 5 1 5 1 2 0 3 3 3 0 5 0 4 0 1 1 1 Table 2 Preparation of Base Agar Matrix Layer 4 After mixing maintain the Base Agar Matrix Layer at 37 C to avoid gelation 5 Dispense 1 mL of Base Agar Matrix Layer into each well of a 6 well sterile plate 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
8. osphere Light Microscope 37 C and boiling water baths Storage Store all components at 4 C until their expiration dates CELL BIOLABS INC ae o gt 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 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 1X Osmotic Lysis Buffer Prepare 1X Osmotic Lysis Buffer by diluting the provided 100X stock 1 100 in sterile cell culture grade water Sterile filter the 1X solution to 0 2 um 10X CytoSelect Agar Matrix Solution Heat the Agar Matrix Solution bottle 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 1X Assay Buffer Prepare 1X Assay Buffer by diluting the provided 10X stock 1 10 in sterile cell culture grade water Sterile filter the 1X solution to 0 2 um Note Only prepare 1X Assay Buffer for colony recovery and re plating since the 10X stock is required for tumor preparation Cell Isolation Protocol must be under sterile conditions The following assay protocol is written for a 6 well plate or 35 mm dish Culture Dish 6 well or 35 mm dish Base Agar Matrix Layer 1000 uL well Cell Suspension Agar 1500 Matrix
9. t 37 C and 5 CO Examine the colony formation under a light microscope IV Recovery of Clonogenic Colonies and Re plating l LOO e H e 10 Add 3 5 mL of 1X Assay Buffer see Preparation of Reagents section to each well Pipette each well 10 12 times to mix thoroughly Transfer the colony suspension mixture 7 mL to a 50 mL conical tube Add 40 mL of serum free culture media to the colony mixture mixing well Pellet the colonies by centrifugation at 1200 rpm for 5 minutes Carefully aspirate the 40 mL of the supernatant Add another 40 mL of serum free culture media to the remaining colony suspension mixing well Place a provided recovery filter into a clean 50 mL conical tube see picture below Carefully pipet the colony suspension step 7 through center of the filter with a steady semi forceful pipet stream Note Clonogenic colonies will be retained on the filter top while single cells will pass through Transfer the filter to another clean 50 mL conical tube 8 Jau BIOLABS INC _ 11 12 13 14 15 16 Wash the retained colonies with 40 mL serum free culture media Add 7 mL of serum free culture media to a clean well of a 6 well plate Quickly transfer the filter to this well submerging the filter bottom into the media Note The filter will tilt within the well but media should fill the inside of the filter chamber Recover the clonogenic colonies by titurating the media inside the fi
10. umor therapy Cell Biolabs CytoSelect Clonogenic Tumor Cell Isolation Kit is designed for the isolation of a small population of colony forming cells from solid tumor samples After digestion of a solid tumor biopsy sample cells are incubated 6 8 days in a proprietary semisolid agar media Once these cells form colonies they are isolated from single cells by a size filter see Isolation Procedure below The viable cells in these colonies are then easily recovered for further culturing and testing FACS protein DNA array analysis cancer vaccine development NOD SCID mouse engraftment is needed to further establish the true stem nature of these isolated cells Each kit provides sufficient quantities to perform five isolations in a 6 well plate or 35 mm dish On average when 500 000 cells from solid tumor are plated after one week culture 50 1000 colonies are expected CELL BIOLABS INC Isolation Procedure E Base Agar Matrix Layer E cei Suspension Agar Matrix Layer e Single Cell Cell Colony Standard Culture Media Patent Pending 5 Solid Tumor Digestion Single Cell Suspension 4 Collagenase Cells in Agar Matrix Incubate 6 8 Days Formation of Cell Colonies isolation of Clonogenic Cells Re plate Isolated Clonogenic Cells Jau BIOLABS INC s Creating Solutions for Life Science Rese ear c h Related Products Oo CO et Oy Oe ee E CBA 100 CytoSelect
11. well CELL BIOLABS INC J AU o gt 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 III allow the plate to warm to room temperature for 30 minutes III Addition of Cell Suspension 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 and CytoSelect Matrix Diluent to 37 C in a water bath Allow at least 30 minutes for the temperature to equilibrate 3 Warm the recovered cell suspension solution Section I step 14 in a 37 C water bath 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 M
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