The dura mater acts as an autonomous B cell niche supporting immune responses during chronic Trypanosoma brucei infection
The dura mater acts as an autonomous B cell niche supporting immune responses during chronic Trypanosoma brucei infection
Whole dura mater was enzymatically digested with Collagenase VIII (1 mg/ml) and DNAse I (1 mg/ml; Sigma) in 1X PBS (HSBB) (Invitrogen) for ~30 minutes at 37 °C and with agitation at 200 r.p.m. Femur samples were cleared from muscle and skin prior to bone marrow isolation. Calvaria were cut into ~1mm pieces. These tissues were then placed on a 0.5 mL Eppendorf tube with a narrow opening at the bottom. This tube was then placed inside a 1.5mL Eppendorf and centrifuge at 800g for 10 minutes to harvest either femur or skull bone marrow. Red blood cells were further lysed from these single cell preparations using ACK lysis buffer (Thermo Fisher, cat. No. A1049201) for 10 minutes on ice, which was then diluted ten times with 1X PBS and cells peletted by centrifugation at 800g for 10 minutes at 4oC Once single cell suspensions were obtained from the whole dura mater, calvaria bone marrow, and femur bone marrow, cells were counted using a hemocytometer, fixed using the EvercodeTM Cell Fixation kit v2 (Parse Bioscience). Cells were pelleted for 10 minutes at 600g and 4oC in 15 ml Falcon tubes pre-coated with 1% BSA to minimise cell adherence and losses. Cell pellets were resuspended in 750 μl of Cell Prefixation buffer, followed by passage through a 40 μm cell strainer to remove cell clumps and addition of 250 μl of Fixation solution. The cells were then incubated for 10 minutes on ice, resuspended in 150 μl of Cell buffer and diluted at a final density of ~500 cells/μl (dura mater) or 10,000 cells/μl (calvaria and femur bone marrow). Libraries were prepared using a split-pool combinatorial barcoding strategy using the Evercode whole transcriptome v2 (Parse Bioscience). The final libraries (~300-500 bp in length) were eluted from SPRI beads using molecular grade water and analysed on a TapeStation.