成年小鼠心脏脂肪细胞分离培养的protocol

PROTOCOL

1.Anesthetize mouse with 150uL heparin (100U/ml) + 0.5mL ketamine/xylazine stock

2.Sacrifice by opening thorax after toe pinch test.

3.Remove and cannulate heart, secure with vascular clip then 6-0 silk. Check cannulation by inflation. Heart should swell.

4.Perfuse heart with 1xPB for 4 min at 4ml/min (16mL)

5.Switch to DB for 3 min at 4ml/min (12mL)

6.Perfuse with DB+CaCl2 for 8 min at 4ml/min (32mL)

7.Heart will be mushy when fully digested and dripping ~16 drops/min

8.Remove from cannula, remove atria and great vessels

9.Place ventricles in sterile 60mm dish with 2.5ml DB

10.Tease ventricles into several pieces. Add 5mL Stopping Buffer (37°C) and pipette several times to release cardiomyocytes from tissue. Solution

should turn cloudy.

11.Transfer cell solution to conical tube with 100µm mesh to filter cells from tissue.

12.Rinse dish with 2.5mL Stopping Buffer and add to tube through mesh. Final volume is 10mL.

13.Let CMs settle 10-20 min at 37°C and plate supernatant in 60mm dish. Let cells settle 2-3 hours in TC incubator. Wash and harvest CFs (plated

cells) or change media and passage.

14.Harvesting CFs:

a.Wash 2x5ml 1xPBS, add 1ml of 1xPBS.

b.Scrape cells into Eppie, cfg twice 3min at 5000rpm at 4°C.

c.Lyse in 350ul RLT+.

SOLUTIONS FOR LANGENDORFF

10x perfusion buffer (stored at 4°C up to 1 month)

1L 500mL

NaCl 70.3g 35.15g

KCl 11g 5.5g

KH2PO4.82g .41g

Na2HPO4.85g .425g

MgSO4●7H2O 3g 1.5g

HEPES**(1M) 100mL 50mL

pH to 7.4 with 1N NaOH ~3mL

mQ H2O to 1L to 500mL

**if HEPES is in powder form, add 13.01mg HEPES to 50mL mQ H2O.

1x perfusion buffer (PB) (make fresh)

250mL 500mL

10x perfusion buffer 25mL 50mL

NaHCO3.0975g .195g

Taurine .9375g 1.875g

BDM (butadionemonoxime) .25g .5g

Glucose .25g .5g

pH to 7.4 with NaOH

mQ H2O to 250mL to 500mL

Filter through 0.2µm

Stopping buffer (7.5mL/heart, make fresh, heat to 37°C before use)

1xPB 9mL

FBS 1mL

100mM CaCl2 1.25µL

Digestion buffer (DB)

1xPB 50mL

320U/mg collagenase II*50mg

100mM CaCl2**15µL

*add immediately before use

**BEFORE adding, remove 12.5mL to fresh tube (for Step 5), THEN add 15µL CaCl2 to remaining

DB. Mix, then put 2.5mL on 60mm plate (for Step 8).

Culture (Fibroblast) medium

MEM 48.5mL

Pen/Strep 0.5mL (1% final)

L-glutamine 0.5mL (1% final)

FBS 50mL (10% final)