PH10005X HMVEC Manual
Contents
1. cells and reagents grants end users a non transferable non exclusive license to use the kit and or its components for internal research use only as described in this manual in particular research use only excludes and without limitation resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis Separate licenses are available for non research use or applications The PrimaPure cells and reagents are not to be used for human diagnostic or included used in any drug intended for human use Care and attention should be exercised in handling the product by following appropriate research lab practices Purchasers may refuse this license by returning the enclosed materials unused By keeping or using the enclosed materials you agree to be bound by the terms of this license The laws of the State of California shall govern the interpretation and enforcement of the terms of this license Page 2 of 2 10190 Telesis Court San Diego CA 92121 Toll Free 888 428 0558 e U S amp Canada 858 457 1919 e www genlantis com 2006 Gelantis and Gene Therapy Sytems Inc2. solution immediately 5 Re cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope It usually takes about 1 minute for the cells to become rounded but still attached to the flask When rounded cells detach by itself without hitting it means the cells are over trypsinized 6 Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached 7 Pipette 5 ml of Trypsin Neutralizing Solution to the flask to inhibit further tryptic activity 8 Transfer the cell suspension from the flask to a 50 ml sterile conical tube 9 Rinse the flask with an additional 5 ml of Trypsin Neutralizing Solution and transfer the solution into the same conical tube 10 Examine the T 25 flask under a microscope If there are gt 20 cells left in the flask repeat Steps 2 9 11 Centrifuge the conical tube at 220 x g for 5 minutes to pellet the cells 12 Aspirate the supernatant from the tube without disturbing the cell pellet 13 Flick the tip of the conical tube with your finger to loosen the cell pellet 14 Resuspend the cells in 5 ml of HMVEC Growth Medium by gently pipetting the cells to break up the clumps 15 Count the cells with a hemocytometer or cell counter Inoculate at 10 000 cells per cm for rapid growth or at 6 000 cells per cm for regular subculturing LICENSE The purchase price paid for the PrimaPure
3. 252 Attachment Factor Solution 100 ml PR123100 GenePORTER 2 Transfection Reagent 0 75 ml 7202007 5 cm2 For example 5 7 5 ml for a T 25 flask or a 60 mm tissue culture dish 15 ml for a T 75 flask or a 100 mm tissue culture dish THAWING AND PLATING HMVEC Remove the cryopreserved vial of HMVEC from the liquid nitrogen storage tank using proper protection for your eyes and hands Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads then re tighten the cap Thaw the cells quickly by placing the lower half of the vial in a 37 C water bath for 1minute Take the vial out of the water bath and wipe dry Decontaminate the vial exterior with 70 alcohol in a sterile Biological Safety Cabinet Remove the vial cap carefully Do not touch the rim of the cap or the vial Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette Be careful not to pipette too vigorously as to cause foaming Pipette 0 5 ml of the cell suspension from the vial into each T 25 flask containing 7 5 ml of HMVEC Growth Medium Cap the flask and rock gently to evenly distribute the cells Place the T 25 flask in a 37 C 5 CO2 humidified incubator Loosen the cap to allow gas exchange For best results do not disturb the culture for 24 hours after inoculation Change to fresh HMVEC Growth Medium after 24 hours or 12 13 14 Human Dermal Microvas
4. PrimaPure Human Dermal Microvascular Endothelial Cells CHMVEC Catalog Description Content Amount PH10005A HMVEC Adult gt 500 000 cells Related Products HMVEC Growth Medium 500 ml US Genlantis A division of Gene Therapy Systems Inc PM112500 PH10005N HMVEC Neonatal gt 500 000 cells HEPES Buffered Saline Solution HBSS 100 ml PR062100 PH10005AK HMVEC Adult Complete System 1 Kit Trpsin EDTA 100 ml PRO70100 PH10005NK HMVEC Neonatal Complete System 1 Kit Trypsin Neutralizing Solution 100 ml PR080100 Each kit contains an ampoule of cryopreserved HMVEC PH10005A or PH10005N 500 ml of HMVEC Cell Growth Medium PM112500 a Subculture Reagent Kit PRO90100k two Extracellular Matrix Protein Coated T 25 flasks PR121252 and Attachment Factor Solution PR123100 INTRODUCTION HMVEC are isolated from normal human neonatal foreskin or adult skin capillaries They are cryopreserved at second passage and can be cultured and propagated at least 16 population doublings They are responsive to cytokine stimulation in the expression of cell adhesion molecules HMVEC express CD36 constitutively while large vessel endothelial cells do not HMVEC is an organ specific endothelial cell system for the study of angiogenesis tumor metastasis2 wound healing inflammation macromolecule transport and drug metabolism There is growing evidence of differe
5. cular Endothelial Cells HMVEC Manual overnight to remove all traces of DMSO Change HMVEC Growth Medium every other day until the cells reach 60 confluent Double the HMVEC Growth Medium volume when the culture is gt 60 confluent or for weekend feedings Subculture the cells when the HMVEC reach 80 confluent Ill Subculturing HDF Cells A PREPARING SUBCULTURE REAGENTS 1 Remove the Subculture Reagent Kit from the 20 C freezer and thaw overnight in a refrigerator 2 Make sure all the subculture reagents are thawed Swirl each bottle gently several times to form homogeneous solutions 3 Store all the subculture reagents at 4 C for future use The activity of Trypsin EDTA Solution will be stable for 2 weeks when stored at 4 C 4 Aliquot Trypsin EDTA solution and store the unused portion at 20 C if only portion of the Trypsin EDTA is needed B PREPARING CULTURE FLASK 1 Swirl the Attachment Factor Solution bottle a few times to form a homogenous solution If the solution is gelled warm up in 56 C water bath for 10 minutes and swirl the bottle to mix the solution 2 Decontaminate the bottle with 70 alcohol in a sterile hood 3 Add 7 5 ml of Attachment Factor Solution to a T 75 flask and rock the flask gently to distribute the solution evenly to cover the whole culture surface 4 Coat the culture ware for 30 minutes at 37 C or 2 hours overnight is also okay at room temperature 5 Remove the Attachment Facto
6. nces in physiology and pharmacology between microvascular endothelial cells and macrovascular endothelial cells 10 MATERIALS AND METHO Preparation for Culturing 1 2 Make sure your Class II Biological Safety Cabinet with HEPA filtered laminar airflow is in proper working condition Clean the Biological Safety Cabinet with 70 alcohol to ensure it is sterile Turn the Biological Safety Cabinet blower on for 10 min before cell culture work Make sure all serological pipettes pipette tips and reagent solutions are sterile Follow the standard sterilization technique and safety rules a Do not pipette with mouth b Always wear gloves and safety glasses when working with human cells even though all the strains have been tested negative for HIV Hepatitis B and Hepatitis C c Handle all cell culture work in a sterile hood Il Culturing HDF Cells A PREPARING CELL CULTURE FLASKS FOR CULTURING HMVEC Take the Extracellular Matrix Protein Coated Flasks and HMVEC Growth Medium from the refrigerator Decontaminate the bottle with 70 alcohol in a sterile hood Prepare two pre coated T 25 flasks for culturing HMVEC by pipetting 7 5 ml of HMVEC Growth Medium to a T 25 flask NOTE Keep the medium to surface area ratio at 1 1 5 ml per Subculture Reagent Kit including100 ml each of HBSS Trpsin EDTA and Trpsin Neutralizing Solution PR090100K Extracellular Matrix Protein Coated T 25 Flasks 2 each PR121
7. r Solution by aspiration in a sterile hood 6 The coated flask can be used immediately or stored at 4 C for up to one month 7 Prepare the coated flask for subculturing by pipetting 15 ml of HMVEC Growth Medium into this coated T 75 flask and wait for seeding NOTE The coating concentration is 1 ml per 10cm surface area of culture ware 2 5 ml for coating a T 25 flask or a 60mm REFERENCES 1 Swerlick R A et al J Invest Dermatol 97 2 190 1991 2 Folkman J et al Nature 288 551 1980 3 Lee K H et al J Invest Dermatol 98 1 79 1992 4 Deryugina E l et al Hybridoma 15 4 279 1996 5 Sepp N T et al J Invest Dermatol 104 2 277 1995 6 Swerlick R A et al J Invest Dermatol 100 1 111S 1993 T Hanasaki K et al J Bio Chem 270 13 7533 1995 8 Pruckler J M et al Pathobiology 61 283 1993 9 Foster C A et al J Dermatol 21 11 847 1994 10 Schnitzer J E et al BBRC 199 1 11 1994 JL060514 Genlantis culture dish 7 5 ml for coating a T 75 flask or a 100mm culture dish C SUBCULTURING HMVEC Trypsinize Cells at Room Temperature Do Not Warm Any Reagents to 37 C 1 Remove the medium from culture flasks by aspiration 2 Wash the monolayer of cells with HBSS and remove the solution by aspiration 3 Pipette 2 5 ml of Trypsin EDTA Solution into the T 25 flask Rock the flask gently to ensure the solution covers all the cells 4 Remove 1 5 ml of the
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