Liver Mononuclear Cell Isolation
From: https://www.sciencedirect.com/science/article/pii/S2211124719313567?via…
Livers from 6-week-old mice were harvested at the naive state (control group) or ninety minutes post 25mg/kg IV Con A injection (experimental group in this study). Single cell suspensions were isolated and enriched for mononuclear cells via Percoll gradient. Briefly, livers were collected in IMDM with GlutaMax (GIBCO) supplemented with 10% FBS, penicillin, streptomycin, and L-glutamine (“complete media”). Livers were minced into small pieces and digested in neat IMDM with 1mg/mL collagenase and 0.2mg/mL DNase at 37°C for 30 minutes with shaking (see above link for vendor lot# information). Cell suspension was further homogenized by flowing through an 18G needle in a 3mL syringe followed by filtering through a 70 μm filter. Following a wash in complete media, mononuclear cells were enriched using a 70%/30% percoll gradient. Cells were washed 2x in complete media and resuspended for downstream applications.
For single cell RNA sequencing library preparation, liver cells are then separated into minireaction “partitions” or Gel bead in emulsion (GEM)s formed by oil micro-droplets, each containing a gel bead and a cell, by the Chromium instrument (10X Genomics). The reaction mixture/emulsion with captured and barcoded mRNAs were removed from the Chromium instrument followed by reverse transcription. The cDNA samples were fragmented and amplified per 10X protocol.
