Adipocyte and SVF isolation from mouse adipose tissue

From LipidomicsWiki

SV-fraction/Adipocyte Isolation
Solutions :
- medium: DMEM (+ pyr + gln, 4,5 g/l gluc), 10 % FBS, Pen/Strep
- collagenase buffer:
NaHCO3 25 mM 84,01 g/mol 1,05 g
KH2PO4 12 mM 136,09 0,81 g
adjust pH 7,4 ad 500 ml, (warm up at 37°C)
MgSO4 1,2 mM 120,37
KCl 4,8 mM 74,55
NaCl 120 mM 58,44
CaCl2*2 H2O 1,4 mM 147,02
Glc 5 mM 180,16
BSA 2,5 %
Pen/Strep filter sterile 1/100

- erythrocyte lysis buffer
NH4Cl 154 mM 0,41 g / 50 ml
KHCO3 10 mM 0,05 g / 50 ml
EDTA 0,1 mM 10 µl / 50 ml (0,5 M stock pH 8,0)

Procedure :
Prepare collagenase solution freshly 2 mg/ml in collagenase buffer
Prepare tube with 2 ul of 0.5M EDTA for blood
Kill animal by CO2
Weight mouse, measure Length of mouse
Quickly open chest cavity and take blood from right ventricle using a 1ml syringe with a 23 ga needle
Measure Glucose Levels (in mM)
Cut right pre ventricle and flush mouse with 5ml of PBS via the left ventricle using a 10 ml syringe and a 27 ga needle
spin blood 8 min at 8000 rpm, transfer plasma into 1.5 ml tube and freeze at -80 °C
Excise subcutaneous and visceral adipose tissue
Mince fat tissue and incubate at 37 °C with mild rocking in collagenase solution for 1h, after 45 min resuspend solution again
Take the following tissues and snap freeze in liquid nitrogen (store at -80 °C):
Liver (large lobe), lung, heart, kidney, (small intestine: stretch intenstine out, measure one forceps length from the stomach and take the part from forceps end to the loop, remove food from small intestine before freezing)
Add same amount of medium to collagenized tissue, resuspend 3 times and transfer to 15 ml falcon
Spin collagenized fat tissue at 1000 rpm for 10 min

SV-fraction
- resuspend S-V fraction in 10 ml medium
- filter through 40 µm net and transfer filtrate to 15 ml falcon
- centrifuge 4 min at 1000 rpm
- resuspend pellet in 5 ml erythrocyte lysis buffer, incubate 5 min at room temperature
- centrifuge 4 min 1000 rpm
- wash with buffer (cold PBS) or medium (for plating) and centrifuge 4 min 1000 rpm

Adipocytes
Take of adipocytes using a 1 ml pipette man with a blue tip that has been cut off
Transfer adipocytes into 8 ml of ice cold PBS
Resuspend 5 times with 10 ml pipette
Pipette onto sieve (180 µm), wash sieve with 2 x 4 ml of PBS (ice)
Take effluent and transfer into 15 ml falcon tube (small adipocytes)
Turn over sieve and wash with 14 ml of PBS (ice), transfer effluent into 15 ml falcon tube (large adipocytes)
Count adipocyte number
Spin adipocytes at 1000 rpm ( g) for 1 min, transfer adipocytes into 1.5 ml tube using a 1ml pipette man with a blue cut of tip