Isolation of Neonatal Rat Myocytes

ADS Buffer:1L 500mL 250mL Conc. (mM)

NaCl 6.8g 3.4g 1.75g 116HEPES 4.76 2.38 1.19 20NaH2PO4 0.12 0.06 0.03 1Glucose 1.0 0.5 0.25 5.5KCl 0.4 0.2 0.1 5.4MgSO4 0.1 0.05 0.025 0.8Phenol red (0.5% in DPBS) 600 µL 300 µL 150 µL

1. Add to 900mL milliQ water2. pH to 7.35±0.05 with 1M NaOH3. Fill up to 1L with mililQ water. Filter with 0.22 µm vacuum filter.4. Store in 4oC

Plating Medium:1L 500mL 250mL

DMEM 660mL 330mL 165mLMedium 199 170 85 42.5Horse serum 100 50 25Fetal calf serum 50 25 12.5Penicillin/streptomycin 5 2.5 1.25HEPES 4.8g 2.4g 1.2g

1. pH to 7.2 with 1M NaOH2. Filter with 0.22 µm vacuum filter3. Store in 4oC

Serum Free Medium:500mL 250mL

DMEM 400mL 200mLM199 100 50Pen/Strep 2.5 1.25HEPES 2.4g 1.2g

pH to 7.2 and filter

Gelatin (1%):1. 4g Gelatin in 400mL milliQ water2. Autoclave to sterilize using the lowest liquid setting3. Store at RT

Collagenase/Pancreatin:

For <11 rats For >12 rats Pancreatin 0.0075g 0.01125gCollagenase 0.026 0.039ADS buffer 50mL 75mL

Make fresh every time, filter sterilize, keep on ice

10mM BrDU:1. Add 0.0015g BrDU to 500mL Plating Media2. Filter using a .22um syringe filter3. Store at -20oC for up to 1 month

List of Reagents and Supplies:

Phenol Red, Sigma cat# P-0290DMEM, Gibco, cat# 11965-118Medium 199, Gibco, cat# 11151-040Horse serum, Gibco, cat# 16050-122NCS, Gibco, cat# 16010-167Pen/Strep, Gibco, cat# 15140-114Gelatin, Sigma, cat# G-9391Collagenase, Worthington, cat# LS004176Pancreatin, Sigma, cat# P3292Trypan blue, Gibco, cat# 15250-0615-Bromo-2-deoxyuridine (BrDU), Sigma cat# B5002-500MG

37oC NCS 12mL37oC Plating Medium (at least 100mL)ADS buffer (at least 125mL)1% Gelatin (at least 50mL)95% EtOH70% EtOHIceForcepsFine scissors, FST cat#14088-10Bags for carcassPaper towels5-6 – 100mm dishes6 – 15 mL Falcon tubes6 – 50mL Falcon tubes60 or 35 mm dishes for plating myocytes

Dissections:1. Decapitate rat pups2. After a midline cut through sternum press down on either side of the cut with gloved

fingers to force the heart out and remove with forceps. Place in 100mm dish with 10mL ADS buffer on ice

3. Repeat for all pups4. Remove hearts to second dish on ice with 10mL ADS 5. Remove large vessels and place in third dish on ice with 10mL ADS6. Chop hearts into 1mm square pieces (roughly in fourths) with fine scissors. Pieces

should be small enough to fit through a 10mL pipette7. Transfer chopped hearts + buffer to 50mL conical tube. Let pieces settle to bottom and

carefully remove media by aspirationDigestion: (protocol for >12 pups)

1. Add 6mL Collagenase/Pancreatin (C/P) solution. Pipette up and down 2-3 times. Incubate 6min 37oC with constant shaking

2. Discard first SN (RBCs, Fibroblasts)3. Add 11-12mL C/P. Pipet up and down 5-6 times. Incubate 18min 37oC with constant

shaking4. Remove SN to 1mL NCS in 15mL falcon tube and keep in hood.5. Repeat steps 3 & 4 five more times (total of 6 digestions)6. After the last digestion centrifuge the 6 15mL tubes @ 1000rpm for 6min (no brake).7. Aspirate the SN and resuspend each pellet in 1mL NCS. 8. Pool all the cells in one 50mL tube. Add 20mL ADS buffer and centrifuge @ 1000rpm

for 6min (no brake)9. Aspirate SN. Resuspend with 4mL plating media and divide into two 100mm dishes with

8mL plating media (if there are >20 hearts use 3 - 100mm dishes). 10. Incubate cells in the dishes for 2hrs @ 37oC. This allows fibroblasts to adhere to dish,

while the myocytes will stay in suspension. 11. During the incubation coat dishes with 1% gelatin enough to cover the bottom of the dish.

Incubate 37oC. Allow the dishes to incubate for at least 10min, although longer is better. Aspirate gelatin and wash 1X with PBS. Coated dishes not used can be stored @ 4oC for two weeks. Warm to RT before use.

12. After 2hr incubation, remove media* and cells to a 50mL tube and centrifuge one last time @ 1000rpm for 6min (no brake). Aspirate SN. Resuspend cells in 4mL plating media. *Do not wash the bottom of the dish. This could loosen fibroblasts from the bottom. Rather, gently swirl the dish to resuspend any myocytes that may have settled.

13. Count cells. Plate 500,000 cells per 60mm dishCounting Cells:

1. Take 50µL of the cell suspension and add it to 50µL Trypan blue. Mix.2. Put 10µL on each side of hemocytometer. Count only the cells that are round or slightly

oval and bright, do not count knobbly or blue cells they will not survive ( dead cells take up trypan blue and will appear blue).

3. Calculate the number of cells:EX: number of cells counted = 100

100x104 X 4 (mL in cell suspension) X 2 (dil. factor) = 800x104 cells total

800x104 cells = 80x105 cells

80x105 / 5x105 = 16 – 60mm dishes4. Typically, 1 heart yields enough cells for 1 - 60mm dish (3 hearts for 1-100mm dish,

1heart for 3-35mm dishes)

Size of dish mL of gelatin mL of plating media

100mm 5mL 10mL 60mm 2mL 3mL 35mm 1mL 1.5mL