PB35205 BPASMC Manual
Contents
1. PrimaPure 34 Bovine Pulmonary Artery Smooth Muscle Cells BPASMC A division of Gene Therapy Systems Inc Catalog Description Content Amount Related Products Catalog PB35205 BPASMC gt 500 000 cells Bovine Smooth Muscle Cell Growth Medium 500 ml PMB311500 PB35205K BPASMC Complete System 1 Kit HEPES Buffered Saline Solution HBSS 100 ml PR062100 Each kit contains an ampoule of cryopreserved BPASMC PB35205 500 Trpsin EDTA 100 ml PRO70100 ml of Bovine Smooth Muscle Cell Growth Medium PMB311500 and a Trypsin Neutralizing Solution 100 ml PR080100 Subculture Reagent Kit PRO90100K 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 1500750 INTRODUCTION Bovine large artery smooth muscle cells are derived from tunica intima and tunica media of healthy fibrous plaque free bovine large arteries They are cryopreserved at second passage and can be cultured and propagated at least 16 population doublings Smooth muscle cells have been found in fatty streaks of early arteriosclerosis Proliferation of the smooth muscle cells is considered a key event in the development of advanced lesions They are an economical alternative suitable for investigations in cardiac disease and arteriosclerosis especially in co culture2. takes about 2 to 5 minutes for the cells to become rounded 5 BPASMC are in growth arrest and smooth muscle a actin is 6 Release the rounded cells from the culture surface by hitting expressed in 10 days 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 peers 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 REFERENCES 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 1 Ross R N 1993 Nature 362 801 2 Stary H C 1983 Arteriosclerosis 3 47 1a 3 McGill H C Jr 1984 Arteriosclerosis 4 443 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 aovern the interpr
3. cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette Be careful not to pipette too Il Culturing BPASMC 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 19 ml of Bovine Smooth Muscle Cell Growth BPASMC Medium 1 Take the Bovine Smooth Muscle Cell Growth Medium from 9 Cap the flask and rock gently to evenly distribute the cells the refrigerator Decontaminate the bottle with 70 alcohol in 10 Place the T 75 flask in a 37 C 5 CO gt humidified incubator a sterile hood 2 Pipette 19 ml of Bovine Smooth Muscle Cell Growth Medium into a T 75 flask 11 Loosen the cap to allow gas exchange For best results do not disturb the culture for 24 hours after inoculation Change to fresh Bovine Smooth Muscle Cell Growth Medium after 24 hours or overnight to remove all traces of DMSO Keep the medium to surface area ratio at 1 ml per 5 cm 12 Change to fresh Bovine Smooth Muscle Cell Growth Medium For example Bovine Pulmonary Artery Smooth Muscle Cells BPASMC Manual every 2 days until the cells reach 60 confluent inhibit further tryptic activity 13 Double the Bovine Smooth Muscle Cell Growth Medium 8 Transfer the cell suspension from the flask to a 50 ml sterile volume when the culture is gt 60 confluent or for weekend conical tube feedings 9 Rinse the flask with an additional 5 ml
4. do 1 to 6 split thereafter 1 Take the Bovine Smooth Muscle Cell Growth Medium from the refrigerator Decontaminate the bottle with 70 alcohol in IV Differentiating BPASMC a sterile hood 2 Pipette 35 ml of Bovine Smooth Muscle Cell Growth Medium A SEEDING BPASMC FOR DIFFERENTIATION to a T 175 flask to be used in Section III C Step 16 1 Seed BPASMC in the desired format at 15 000 per cm2 Follow instructions in Section IV C C SUBCULTURING BPASMC 2 Change to Bovine Smooth Muscle Differentiation Medium the Trypsinize Cells at Room Temperature Do Not Warm Any next day Reagents to 37 C B DIFFERENTIATING BPASMC TO EXPRESS CONTRACTILE 1 Remove the medium from culture flasks by aspiration PROTEIN 2 Wash the monolayer of cells with HBSS and remove the 1 Remove growth medium from culture tissue ware by solution by aspiration aspiration Do not allow cells to dry during medium changes 3 Pipette 5 ml of Trypsin EDTA Solution into the T 75 flask 2 Add the appropriate volume of Bovine Smooth Muscle Rock the flask gently to ensure the solution covers all the Differentiation Medium cells 3 Incubate cell in a 37 C 5 CO2 humidified incubator in the 4 Remove 4 ml of the solution immediately Bovine Smooth Muscle Differentiation Medium 5 Re cap the flask tightly and monitor the trypsinization progress 4 Change to fresh Bovine Smooth Muscle Differentiation at room temperature under an inverted microscope It usually Medium every other day
5. etation and enforcement of the terms of this license MV060530 Genlantis 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
6. of Trypsin Neutralizing 14 Subculture the cells when the BPASMC reach 80 confluent Solution and transfer the solution into the same conical tube 10 Examine the T 75 flask under a microscope If there are gt 20 Ill Subculturing BPASMC cells left in the flask repeat Steps 2 9 11 Centrifuge the conical tube at 220 x g for 5 minutes to pellet A PREPARING SUBCULTURE REAGENTS the cells 12 Aspirate the supernatant from the tube without disturbing the 1 Remove the Subculture Reagent Kit from the 20 C freezer cell pellet and thaw overnight in a refrigerator 13 Flick the tip of the conical tube with your finger to loosen the 2 Make sure all the subculture reagents are thawed Swirl each cell pellet bottle gently several times to form homogeneous solutions 14 Resuspend the cells in 2 ml of Bovine Smooth Muscle Cell 3 Store all the subculture reagents at 4 C for future use The Growth Medium by gently pipetting the cells to break up the activity of Trypsin EDTA Solution will be stable for 2 weeks clumps when stored at 4 C 15 Count the cells with a hemocytometer or cell counter 4 Aliquot Trypsin EDTA solution and store the unused portion at Inoculate at 15 000 cells per cm for rapid growth or at 10 000 20 C if only portion of the Trypsin EDTA is needed cells per cm for regular subculturing 16 Transfer the 2 ml cell suspension to a T 175 flask containing B PREPARING CULTURE FLASK 35 ml of Bovine Smooth Muscle Cell Growth Medium and
7. with species matched bovine arterial endothelial cells MATERIALS AND METHODS F 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 BPASMC filtered laminar airflow is in proper working condition 1 Remove the cryopreserved vial of BPASMC 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 d that may be trapped in the threads then re tighten the cap Make sure all serological pipettes pipette tips and reagent 3 Thaw the cells quickly by placing the lower half of the vial in a seUlibisale stenig menai 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 o gt Boney pene wmo 5 Decontaminate the vial exterior with 70 alcohol in a sterile b Always wear gloves and safety glasses when working Biological Safety Cabinet Mal puan pa even though all the USDA has 6 Remove the vial cap carefully Do not touch the rim of the cap inspected all the animals orthe vial E AO noo 7 Resuspend the
Download Pdf Manuals
Related Search