Isolation of Neonatal Rat Myocytes

ADS Buffer:
1L 500mL 250mL Conc. (mM)
NaCl 6.8g 3.4g 1.75g 116
HEPES 4.76 2.38 1.19 20
NaH2PO4 0.12 0.06 0.03 1
Glucose 1.0 0.5 0.25 5.5
KCl 0.4 0.2 0.1 5.4
MgSO4 0.1 0.05 0.025 0.8
Phenol red (0.5% in DPBS) 600 µL 300 µL 150 µL

1. Add to 900mL milliQ water

2. pH to 7.35±0.05 with 1M NaOH
3. 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 165mL
Medium 199 170 85 42.5
Horse serum 100 50 25
Fetal calf serum 50 25 12.5
Penicillin/streptomycin 5 2.5 1.25
HEPES 4.8g 2.4g 1.2g

1. pH to 7.2 with 1M NaOH

2. Filter with 0.22 µm vacuum filter
3. Store in 4oC

Serum Free Medium:

500mL 250mL
DMEM 400mL 200mL
M199 100 50
Pen/Strep 2.5 1.25
HEPES 2.4g 1.2g

pH to 7.2 and filter

Gelatin (1%):
1. 4g Gelatin in 400mL milliQ water
2. Autoclave to sterilize using the lowest liquid setting
3. Store at RT

Collagenase/Pancreatin:

For <11 rats For >12 rats

Pancreatin 0.0075g 0.01125g
Collagenase 0.026 0.039
ADS buffer 50mL 75mL

Make fresh every time, filter sterilize, keep on ice

10mM BrDU:
1. Add 0.0015g BrDU to 500mL Plating Media
2. Filter using a .22um syringe filter
3. Store at -20oC for up to 1 month
List of Reagents and Supplies:

Phenol Red, Sigma cat# P-0290

DMEM, Gibco, cat# 11965-118
Medium 199, Gibco, cat# 11151-040
Horse serum, Gibco, cat# 16050-122
NCS, Gibco, cat# 16010-167
Pen/Strep, Gibco, cat# 15140-114
Gelatin, Sigma, cat# G-9391
Collagenase, Worthington, cat# LS004176
Pancreatin, Sigma, cat# P3292
Trypan blue, Gibco, cat# 15250-061
5-Bromo-2-deoxyuridine (BrDU), Sigma cat# B5002-500MG

37oC NCS 12mL

37oC Plating Medium (at least 100mL)
ADS buffer (at least 125mL)
1% Gelatin (at least 50mL)
95% EtOH
70% EtOH
Ice
Forceps
Fine scissors, FST cat#14088-10
Bags for carcass
Paper towels
5-6 – 100mm dishes
6 – 15 mL Falcon tubes
6 – 50mL Falcon tubes
60 or 35 mm dishes for plating myocytes
Dissections:
1. Decapitate rat pups
2. 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 pups
4. Remove hearts to second dish on ice with 10mL ADS
5. Remove large vessels and place in third dish on ice with 10mL ADS
6. Chop hearts into 1mm square pieces (roughly in fourths) with fine scissors. Pieces
should be small enough to fit through a 10mL pipette
7. Transfer chopped hearts + buffer to 50mL conical tube. Let pieces settle to bottom and
carefully remove media by aspiration
Digestion: (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
shaking
4. 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 dish
Counting 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 dishes

4. 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