Preparation of mouse liver non-parenchymal cell fraction
- Sacrifice mouse
- In vivo perfusion with 5-10 ml of PBS from the IVC follows by 5-10ml of Liver Digest Medium (Gibco)
- Harvest the liver without including the gall bladder and transfer to a tube with (Williams’E medium + 10% FCS).
- Cut the liver into fine pieces and incubate in Liver Digest Medium at 37C for 20 mins
- Mesh the liver more either with mechanical chopping with a scalpel or Miltenyi GentleMACS.
- Pass the mesh through 100um cell strainer and then top up with 25ml of Williams’E medium + 10% FCS
- Spin tubes at 50G for 5 mins with acceleration =4 deceleration=4
- Transfer supernatant to a new 50 ml falcon while passing through a 40um strainer, discard the pellet.
- Spin tubes at 50G for 5 mins with acceleration =4 deceleration=4
- Transfer supernatant to a new 50 ml falcon while passing through a 40um strainer, discard the pellet.
- Spin tubes at 300G for 5 mins with max acceleration max deceleration
- Keep pellet, discard supernatant
- Incubate with red cell lysis buffer for 2 mins on ice.
- Wash with 10 ml of PBS at 300G
- Spin tubes at 300G for 5 mins with max acceleration max deceleration
- The resulting pellet will be the non-parenchymal fraction of the liver. Can either plate or continue with FACS.
FACS of mouse liver non-parenchymal cell fraction
- Block with either Fc Block or neat FCS for 10 mins at room temperature
- Spin at 300G for 5 mins, discard supernatant
- Incubate with antibody mix for 30mins – 1 hour on ice. ~50-100 ul per sample
Antibody Mix
Manufacturer Antibody Clone Dilution
eBiosciences CD45 PE 30-F11 1/100
eBiosciences CD31 PE 390 1/100
eBiosciences Ter119 PE TER119 1/100
eBiosciences EpCAM APC G8.8 1/200
eBiosciences CD133 FITC 13A4 1/50
Biolegend CD24 PeCy7 M1/69 1/200
- Wash with FACS Buffer (PBS +2%FCS)
- Spin at 300G for 5 mins, discard supernatant.
- Resuspend samples in 300ul of FACS buffer and transfer to FACS tube with 40um cell strainer cap and sort for CD45-/CD31-/Ter119-/EpCAM+/CD24+/CD133+
- Before sorting, add life dead marker to exclude dead cells, DAPI/7AAD
Culturing mouse liver NPCs / HPCs
- Coat plates with 1 mg/ml Rat Tail Collagen 1 (Sigma)
- Leave in incubator for approximately 4 hours to dry
- Plate cells with liver expansion medium
HPCs Expansion Medium Recipe (Williams’ E + 10% FCS)
Chemicals required Stock concentration Dilution Preparation
17.6 mM NaHCO3
20mM HEPES pH 7.5
10 mM Nicotinamide
1mM Sodium Pyruvate
1X ITS
100nM Dexamethasone
0.2 mM Ascorbic Acid
14mM glucose 1.4 M
10ng/mL IL-6 10 ug/mL
10ng/mL HGF 20ug/mL
10ng/mL EGF 100ug/mL
- Wait for colony to form
- After colony formation, change medium every 2-3 days
- Try and prepare medium relatively fresh, in 5ml aliquots (or smaller)
Passaging primary mouse HPCs
- Incubate with diluted trypsin until cells start to detach (might take a while)
- Flush colony with Williams’E + 10%FCS
- Collect cells and spin at 300G for 5 mins.
- Replate cells on collagen coated plates
Diluted Trypsin (Tsuchiya et al, Gastroenterology 2005):
0.25% Trypsin no EDTA
20% Knockout Serum Replacement
1mM CaCl2