Metzger Lab Protocol Book EF May 2003

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1 I. Preparation for cell isolation: A. Make sure the following items are autoclaved: 1. Glassware* (1) 100 ml beaker (3) wide mouthed 1 liter bottles (1) 100 mm glass petri dish (1) 60 mm sigma coated petri dish * Note: cover with foil or autoclave tubing Based on UCSF Protocol MOUSE CARDIAC MYOCYTE ISOLATION PROTOCOL FOR PRIMARY CULTURE July Surgical instruments** scissors dissecting scissors (2) fine tipped forceps blunt forceps cannula ** Note: Place all the surgical instruments in surgical autoclave tubing B. Prepare solutions. 1. Sterile dd H 2 0 Filter through a.2 um filter into an autoclaved 1L bottle 2. Sterile dd H 2 0 with 10 ml P/S. Use the same.2 mm filter as above into another autoclaved 1L bottle % Ethanol: dilute 2.2 L of 95% ethanol with.8 L of dd H l Buffer Solution ph 7.4 (preferably made the morning of isolation): Reagent Molarity of Stock Solution (M) Grams / 1 L Stock Solution Final Molarity (mm) Quantity / 1 L Buffer NaCl ml KCl ml MgSO ml NaH2PO ml KH2PO ml HEPES Fresh ml BDM Fresh g NaHCO3 Fresh g Taurine Fresh g Glucose Fresh g 5. Collagenase Stopping Buffer: Reagent Quantity Final concentration Buffer Solution 30 ml Bovine Serum Albumin 0.75 g 2.5% 1 M CaCl 2 stock 3.0 ul 0.1 mm

2 6. Culture Media: (preferably made the morning of isolation) * Only open MEM in the hood. Mark the Invitrogen container with the date opened and that it is only to be opened in the hood. Reagent Quantity Final Concentration MEM 150 ml Bovine Serum Albumin 0.3 g 0.2% Penicillin 1.5 ml 100 units/ml Glutamine 1.5 ml 2 mm 2,3 Butanedione monoxime 0.45 g 20 mm ITS 150 ul I 1ug/ml; T 0.55 ug/ml; S 0.5 ng/ml NaHCO g 4 mm HEPES 1.5 ml 10 mm 7. Plating Media: Reagent Culture Media Bovine Calf Serum Quantity 15 ml 0.75 ml 8. CaCl2: 147 g in 1 L dd H20 9. Laminin: 1 ml thawed laminin (from enzyme core) + 24 ml sterile Phosphate Buffered Saline II. Setup: A. Place plating medium in incubator B. Prepare collagenase solution 175 mg Collagenase Type 2 Lot 42D mg Protease XIV Lot 99H mg Dnase I grade II 100 ml buffer solution C. Wash out the perfusion apparatus ml Ethanol ml water ml water with P/S 4. When viability and yield begin to decrease or every 2-3 months, clean the apparatus. First run dilute bleach through the apparatus and rinse with dd H 2 0. Then break down the apparatus and wash with 7x soap and pipe cleaners, rinse extensively with dd H 2 0 before setting back up. Use all new tubing each time. D. Flush system with Buffer solution being sure to clear all bubbles. Have collagenase free buffer in one syringe and buffer with collagenase in the other. E. Turn on water bath F. Arrange dissecting area under magnifying glass 2 blue pads 2 petri dishes with stock solution 1 10 cc syringe containing buffer solution with cannula in one petri dish (place the cannula on the syringe and mount such that the cannula is just off the floor of the dish) Pre-tie a piece of surgical silk around the cannula all scissors and forceps placed in a clean plastic petri dish Spray bottle of ethanol sample buffer (for sample collection) G. Collect mouse and heparinize the mouse with.1 cc heparin; wait for 15 minutes.

3 III. Collecting the heart: A. Anesthetize the mouse with.1 cc sodium pentobarbital and begin checking for reflexes every 2 minutes (check by pinching feet with forceps). B. Spray abdomen with ethanol C. Cut open thorax and remove the lungs and excess fat and connective tissue. D. Cut out the heart above aortic arch trying to leave > 2mm of the aorta on the heart. E. Put the heart directly into one of the petri dishes containing buffer solution and find the aorta and adjust the length to approximately 2 mm. F. Gently slide the aorta over the cannula so the bevel is just visible inside the aorta. If necessary, use a vascular clamp to hold the aorta in place while you secure it with a surgical tie. G. Remove the vascular clamp and test for perfusion by gently flushing the heart with stock solution from the syringe. The heart should swell slightly and blood should be expelled. IV. Perfusion: A. Start slow drip on perfusion apparatus from the syringe with collagenase free buffer and transfer the cannula with heart to perfusion apparatus. Be sure the cannula fills with solution so as to avoid forcing air through the heart. Once the heart is secure turn on flow completely. B. Measure the flow rate. 3 ml/ min is optimal. C. Once the heart has been perfused with the collagenase free buffer for 4 minutes switch to the collagenase solution for 2 minutes. D. Add 2.5 ul of 1 M Ca to 50 ml (35 ml in syringe + 15 ml in tubing) of collagenase solution and continue to perfuse 8 minutes. V. Cell isolation: E. Remove the heart from the cannula and place it in a small, sigma coated petri dish with 5 ml of the digest solution from the syringe. F. Remove the atrium, aorta, cannula and thread from the ventricles using the dissecting scissors and needlenosed forceps. G. Cut the heart in half with scissors and tease the tissue apart with fine forceps until only small chunks are left H. With the tweezers, remove a small portion of the tissue for sample collection in the 350 ul of sample buffer and put that into the 80 freezer. I. Gently triturate the remaining tissue with a sterile 5 ml plastic pipette 5 6 times and then transfer the contents of the dish to a 15 ml conical tube. J. Gently triturate the remaining tissue with a pasteur pipette 5 6 times and then add 5 ml of stopping buffer to cease enzyme activity. K. Allow cells to settle into a loose pellet for 3 minutes. During this time take a small sample on a slide and check at the microscope for cell viability. L. Transfer the supernatant to a new 15 ml tube and add 10 ml of stopping buffer to the original pellet. M. Centrifuge the supernatant for 10 seconds at speed 4. N. Discard the supernatant and combine the pellets in the 10 ml of stopping buffer. VI. Calcium add back: A. Resuspend the cells in the 10 ml of stopping buffer, transfer to the sigma coated petri dish, and let settle for 5 minutes. B. Add 1 ul of 1 M CaCl 2 4 times at 5 minute intervals. Let rest 5 minutes after final addition of calcium. VII. Washing the cells: A. Transfer the contents of the petri dish to a 15 ml conical tube and let settle for 2 minutes. B. Transfer the supernatant to a second 15 ml conical tube and add 5 ml of plating media to the pellet in the first 15 ml conical tube.

4 C. Centrifuge the contents of the second tube (the supernatant) 10 seconds at speed 4 and discard the supernatant. D. Discard the supernatant and combine pellets in 5 ml of plating medium VIII. Count Cells: A. Place small drops on the hemocytometer so that the solution and cells can spread to where the grids are. B. Using the microscope, find the grids and count the rod shaped and dead cells from 9 fields. C. Calculate the percentage of rod shaped cells and the average concentration of cells (concentration = # of cells in 1 field x 10 4 in 1 ml of solution). Remember to multiply by 5 (for the 5 ml of cells in solution). A good prep yields approximately 1,000,000 rod shaped cells at 80 % viability. D. Add plating media to the collected cells to bring the concentration to 100,000 cells / 1 ml and place in incubator. IX. Plating: A. Place 1 coverslip in each well of a 6 well plate. UV uncovered for 10 minutes. B. Coat each coverslip with 100 ul laminin (40mg/ml). UV uncovered for 10 minutes. C. Replace the lids for the plates until ready to proceed. D. Remove excess laminin from coverslips by aspirating. E. Resuspend the cells in the plating media. F. Carefully place 200 ul cell suspension / coverslip. Do not allow solution to roll off the coverslip. G. Place plates in incubator carefully so as to not disturb cells. H. Incubate for 2 hours, remove serum containing media by aspirating from the edge of the coverslip. At this point you can infect cells with adenovirus or add back 2mls of culture media. If infecting wait one hour after infection, then add 2 ml culture media to each well.

5 Ordering Information Reagent Description Quantity Storage Catalog Info 2,3-butanedione Solid 25 g Room Sigma B0753 monoxime Fetal Bovine Serum liquid 500 ml 5 ml aliquots in -20 in 7727 Atlanta Biologicals S11550 Bovine Serum Solid 50 g 10% Stock Solution: 5ml Sigma A8806 Albumin aliquots in 20 in 7727 CaCl2 solid 500 g Stock Solution: Room Sigma C3881 Collagenase Type 2 Lot # 42D5572 Solid 1 g refrigerator desicator Worthington bulk reserves Dnase I grade 2 Solid 100 mg Refrigerator desicator From Core Roche Dulbecco s Liquid 500 ml Cold room Gibco Phosphate Buffered Saline Glucose Solid 250 g Room Sigma G7528 HEPES Liquid 100 ml Refrigerator 7727 Invitrogen ITS Liquid 5 ml refrigerator Sigma I 3146 KCl Solid 250 g Stock Solution: Room Sigma P5405 KH2PO4 Solid 100 g Stock Solution: Room Sigma P5655 L Glutamine Liquid 100 ml 5 ml aliquots in 20 in Gibco MEM Liquid 500 ml cold room Gibco MgSO4-7H2O Solid 500 g Stock Solution: Room Sigma M1880 Mouse Laminin Liquid 1 ml Diluted in 24 ml PBS, 1 ml aliquots in 80 freezer From Core Invitrogen NaCl Solid 500 g Stock solution: Room Sigma S9625 NaH2PO4 Solid 100 g Stock Solution: Room Sigma S0751 NaHCO3 Solid 500 g Room Sigma S5761 Pen/Strep Liquid 100 ml -20 room 7731, 5 ml Gibco aliquots Protease XIV Lot 99H0709 Solid 1 g - 20 Freezer in bucket From Core Sigma P5147 Taurine Solid 100 g Room Sigma I0625