成年小鼠心脏脂肪细胞分离培养的protocol
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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)