GSM5708984: HTO_Meningeals cells of 2 PBS & 2 LPS mice; Mus musculus; RNA-Seq
Hundred μl of LPS (Sigma, 0.1 mg/ml)  were injected intraveinously via the retro-orbital vein. Anesthetized mice received an intracardiac perfusion with PBS to remove contaminating erythrocytes. Leukocytes were isolated from the meninges using forceps to gently separate them from the underside of skull cap (the same method used to prepare meningeal whole mounts above). This was followed by enzymatic digestion in RPMI containing 2.5 mg/ml collagenase D (Roche) + 0.1 mg/ml DNase (Roche) for 30 min at 37 °C with gentle shaking every 10 min. Leukocytes were also isolated from the brain and the spleen as described. Following digestion, supernatants were isolated, washed, and finally passed through a 35-μm pore cell strainer. After this step, meningeal cells were used for staining. Cells from the brain, however, were resuspended in 40% Percoll (GE Healthcare) gradient in PBS1X and centrifuged to remove myelin and debris from the preparation. Afterwards, brain leukocytes were stained comparably to meningeal cells. Spleen samples were similarly digested. After straining, spleen leukocytes were treated with RBC1X to eliminate red blood cells, then washed and 1:20 of total cells were isolated for cell staining.