PH35005A HCASMC Manual
Contents
1. 14 Subculture the cells when the HCASMC reach 80 confluent Ill Subculturing HCASMC 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 PREPARING CULTURE FLASK 1 Take the Smooth Muscle Cell Growth Medium from the refrigerator Decontaminate the bottle with 70 alcohol in a sterile hood 2 Pipette 30 ml of Smooth Muscle Cell Growth Medium to a T 175 flask to be used in Section Ill C Step 15 C SUBCULTURING HCASMC 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 5 ml of Trypsin EDTA Solution into the T 75 flask Rock the flask gently to ensure the solution covers all the cells 4 Remove 4 ml of the solution immediately 5 Re cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope It usually takes about 2 to 5 minutes for the cells t2. PrimaPure 34 Human Coronary Artery Smooth Muscle Cells CHCASMC A division of Gene Therapy Systems Inc Catalog Description Content Amount Related Products Catalog PH35005A HCASMC gt 500 000 cells Smooth Muscle Cell Growth Medium 500 ml PM311500 PH35005AK HCASMC Complete System 1 Kit HEPES Buffered Saline Solution HBSS 100 ml PR062100 Each kit contains an ampoule of cryopreserved HCASMC PP35005 500 ml Trpsin EDTA 100 ml PR070100 of Smooth Muscle Cell Growth Medium PM311500 and a Subculture Trypsin Neutralizing Solution 100 ml PR080100 Reagent Kit PRO901 00K Subculture Reagent Kit including100 ml each of HBSS PR090100K Trpsin EDTA and Trpsin Neutralizing Solution GenePORTER 2 Transfection Reagent 0 75 ml 7202007 GeneSilencer siRNA Transfection Reagent 200 reactions T500750 INTRODUCTION Human Coronary Artery Smooth Muscle Cells HCASMC are derived from the tunica intima and tunica media of normal human fibrous plaque free coronary arteries They are cryopreserved at second passage and can be cultured and propagated at least 16 population doublings Smooth muscle cells are present in various amounts in early fatty streaks and they are the predominant type of cells in fibrous plaques 2 Atherosclerosis of the coronary arteries can cause angina pectoris and heart attack Intimal smooth muscle proliferation plays a key event in the development of atherosclerosis HCASMC is
3. an ideal cellular model for the investigations of coronary artery diseases such as alteration of smooth muscle cell differentiation in atherosclerosis MATERIALS AND METHODS LP tion for Culturi 5 ml for a T 25 flask or a 60 mm tissue culture dish Separation Ob Mound 15 ml for a T 75 flask or a 100 mm tissue culture dish 1 Make sure your Class II Biological Safety Cabinet with HEPA B THAWING AND PLATING HCASMC filtered laminar airflow is in proper working condition 1 Remove the cryopreserved vial of HCASMC from the liquid 2 Clean the Biological Safety Cabinet with 70 alcohol to nitrogen storage tank using proper protection for r eye ensure it is sterile gen storage tank using proper protection for your eyes and hands 3 Turn the Biological Safety Cabinet blower on for 10 min 2 Turn the vial cap a quarter turn to release any liquid nitrogen before cell culture work that may be trapped in the threads then re tighten the cap i a sure all serological pipettes pipette tips and reagent 3 Thaw the cells quickly by placing the lower half of the vial in a solutions are sterile POTAS 37 C water bath for 1 minute 5 Follow the standard sterilization technique and safety rules 4 Take the vial out of the water bath and wipe dry a Do not pipette with mouth b Always wear gloves and safely glasses w hen working 5 Decontaminate the vial exterior with 70 alcohol in a sterile with human cells even though all the strains have be
4. en Biological Safety Cabinet tested negative for HIV Hepatitis and Hepatitis C 6 Remove the vial cap carefully Do not touch the rim of the cap c Handle all cell culture work in a sterile hood or the vial 7 Resuspend the cells in the vial by gently pipetting the cells 5 Il Culturing HEASMC times with a 2 ml pipette Be careful not to pipette too vigorously as to cause foaming A PREPARING CELL CULTURE FLASKS FOR CULTURING 8 Pipette the cell suspension 1ml from the vial into the T 75 HCASMC flask containing 15 ml of Smooth Muscle Cell Growth Medium 1 Take the Smooth Muscle Cell Growth Medium from the 9 Cap the flask and rock gently to evenly distribute the cells refrigerator Decontaminate the bottle with 70 alcohol in a 10 Place the T 75 flask in a 37 C 5 CO2 humidified incubator sterile hood a Loosen the cap to allow gas exchange For best results do 2 Pipette 15 ml of Smooth Muscle Cell Growth Medium into a not disturb the culture for 24 hours after inoculation T 75 flask 11 Change to fresh Smooth Muscle Growth Medium after 24 X hours or overnight to remove all traces of DMSO Keep the medium to surface area ratio at 1 ml per 5 cm 12 Change Smooth Muscle Cell Growth Medium every other day For example Human Coronary Artery Smooth Muscle Cells HCASMC Manual until the cells reach 60 confluent 13 Double the Smooth Muscle Cell Growth Medium volume when the culture is gt 60 confluent or for weekend feedings
5. o become rounded The cells may not be completely round during trypsinization and some cells may maintain some processes even though they are loosened from the culture surface 6 Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells REFERENCES 1 Ross R N Nature 362 801 1993 2 McGill H C Jr Arteriosclerosis 4 443 1984 3 Jonasson L et al Artherosclerosis 6 2 131 1986 4 Owens G K Atherosclerosis and Coronary Artery Disease Chap3 401 1996 5 Fager G et al In Vitro Cell Biol 25 6 511 1989 JL060514 Genlantis 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 75 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 Smooth Muscle Cell Growth Medium by gently pipetting the cells to break up the clumps 15 Count the cells with a hemocy
6. tometer or cell counter Inoculate at 10 000 cells per cm for rapid growth or at 6 000 cells per cm for regular subculturing IV DIFFERENTIATING HCASMC A SEEDING HCASMC FOR DIFFERENTIATION Seed HCASMC in the desired format at 15 000 per cm2 Follow instructions in Section IV C 2 Change to Smooth Muscle Differentiation Medium the next day x B DIFFERENTIATING HCASMC TO EXPRESS CONTRACTILE PROTEIN 1 Remove growth medium from culture tissue ware by aspiration Do not allow cells to dry during medium changes 2 Add the appropriate volume of Smooth Muscle Differentiation Medium 3 Incubate cell in a 37 C 5 CO2 humidified incubator in the Smooth Muscle Differentiation Medium 4 Change to fresh Smooth Muscle Differentiation Medium every other day 5 HCASMC are in growth arrest and smooth muscle actin is expressed in 10 days LICENSE The purchase price paid for the PrimaPure 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 dr
7. ug 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 Inc
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