PH35205A HPASMC Manual
Contents
1. PrimaPure 34 Human Pulmonary Artery Smooth Muscle Cells HPASMC A division of Gene Therapy Systems Inc Catalog Description Content Amount Related Products Catalog PH35205A HPASMC Adult gt 500 000 cells Smooth Muscle Cell Growth Medium 500 ml PM311500 PH35205AK HPASMC Adult Complete System 1 Kit HEPES Buffered Saline Solution HBSS 100 ml PR062100 Each kit contains an ampoule of cryopreserved HPASMC PH35205A 500 Trpsin EDTA 100 ml PRO70100 ml of Smooth Muscle Cell Growth Medium PM311500 and a Subculture Trypsin Neutralizing Solution 100 ml PR080100 Reagent Kit PRO90100K Subculture Reagent Kit including100 ml each of HBSS PRO90100K Trpsin EDTA and Trpsin Neutralizing Solution GenePORTER 2 Transfection Reagent 0 75 ml 7202007 GeneSilencer siRNA Transfection Reagent 200 reactions T500750 INTRODUCTION Human Pulmonary Artery Smooth Muscle Cells HPASMC are derived from tunica intima and tunica media of normal human fibrous plaque free pulmonary arteries They are cryopreserved at second passage and can be cultured and propagated at least 16 population doublings HPASMC are well suited for the study of pulmonary vascular disorders associated liver diseases and connective tissue diseases Phenotypic changes of smooth muscle cells have been found to be related to smooth muscle cell migration and intimal proliferation Switches in smooth muscle cell phenotype lead to2. pulmonary hypertension and eventually heart and lung disorders MATERIALS AND METHODS 5 ml for a T 25 flask or a 60 mm tissue culture dish Preparation for Culturing 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 HPASMC filtered laminar airflow is in proper working condition 1 Remove the cryopreserved vial of HPASMC from the liquid 2 Clean the Biological Safety Cabinet with 70 alcohol to nitrogen storage tank using proper protection for your eyes ensure it is sterile g g g proper p y y 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 A Make 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 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 5 Decontaminate the vial exterior with 70 alcohol in a sterile b Always wear gloves and safety glasses when working Biological Safety Cabinet with human cells even though all the strains have been 6 Remove the vial cap carefully Do not touch the rim of the cap tested negative for HIV Hepatiti
3. CULTURE 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 HPASMC 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 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 to become rounded The cells may not be completely round during trypsinization and some cells may maintain s
4. ised 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
5. ome processes even though they are loosened from the culture surface 6 Release the rounded cells from the culture surface by hitting mn REFERENCES 1 Rabinovitch M Moss Heart Disease in Children Infants and Adolescents Williams and Wilkins Baltimore p 856 1989 Levine O R et al J Pediatr 83 964 1973 3 Nair S S et al Arch Intern Med 140 109 1980 4 Rabinovitch M Toxicologic Pathology Vol 19 No 4 Pt 1 pp 458 468 1991 JL060514 Genlantis 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 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 2 ml of Smooth Muscle Cell 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 fo
6. r rapid growth or at 6 000 cells per cm for regular subculturing lV Differentiating HPASMC A SEEDING HPASMC FOR DIFFERENTIATION Seed HPASMC 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 B DIFFERENTIATING HPASMC 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 HPASMC are in growth arrest and smooth muscle c 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 drug intended for human use Care and attention should be exerc
7. s B and Hepatitis C or the vial c Handle all cell culture work in a sterile hood 7 Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette Be careful not to pipette too ll Culturing HPASMC vigorously as to cause foaming 8 Pipette the cell suspension 1ml from the vial into the T 75 A PREPARING CELL CULTURE FLASKS FOR CULTURING flask containing 15 ml of Smooth Muscle Cell Growth Medium HPASMC 9 Cap the flask and rock gently to evenly distribute the cells 1 Take the Smooth Muscle Cell Growth Medium from the 10 Place the T 75 flask in a 37 C 5 CO2 humidified incubator refrigerator Decontaminate the bottle with 70 alcohol in a Loosen the cap to allow gas exchange For best results do sterile hood not disturb the culture for 24 hours after inoculation 2 Pipette 15 ml of Smooth Muscle Cell Growth Medium into a 11 Change to fresh Smooth Muscle Cell Growth Medium after 24 T 75 flask hours or overnight to remove all traces of DMSO 12 Change Smooth Muscle Cell Growth Medium every other day Keep the medium to surface area ratio at 1 ml per 5 cm2 until the cells reach 60 confluent For example 13 Double the Smooth Muscle Cell Growth Medium volume when Human Pulmonary Artery Smooth Muscle Cells HPASMC Manual the culture is gt 60 confluent or for weekend feedings 14 Subculture the cells when the HPASMC culture reaches 80 confluent Subculturing HPASMC PREPARING SUB
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