Summit software version 4

Peritoneal cells were subjected to flow cytometry analysis using the antibodies CD19-Alexa Fluor 647 (rat anti-mouse IgG2a, clone: 6D5; BioLegend, San Diego, California, USA), CD5-PerCP (rat anti-mouse IgG2a, clone: 53–7.3; BD Pharmingen, San Jose, California, USA), and CD11b-PE (rat anti-mouse IgG2b, clone: M1/70; BD Pharmingen, San Jose, California, USA). Measurement was made using a CyAn ADP flow cytometer (Dako, Glostrup, Denmark). B-1a cells were characterized as CD19⁺CD5⁺CD11b⁺, B-1b cells as CD19⁺CD5^-CD11b⁺, and B-2 cells as CD19⁺CD5^-CD11b^-, using the CyAn ADP flow cytometer and Summit software version 4.3 (Dako, Glostrup, Denmark). Rat IgG2a FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) and rat IgG2b FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) were used as the isotype controls.

BAL cells were collected as described previously. Cells were fixed in a 1‐step fix/lyse solution (eBiosciences, CA), washed twice in PBS, and the pellets were suspended in staining buffer. BAL cells were analyzed for the mEGFP and mTom fluorescence with Dako CyAn (Beckman Coulter, Inc., CA). Flow cytometric data were analyzed using Summit software Version 4.3 (Dako, CA).

Cell size, granularity, and fluorescence intensity were analyzed per brain region (n = 3) with a MoFlo fluorescent activated cell sorter (Beckman Coulter, Mijdrecht, Netherlands). Approximately 2x10⁴ events were counted and subsequently analyzed using Summit software version 4.3 (DAKO) to determine gates before cells were sorted. Microglia were defined as the percentage of all living, Sytox green negative, cells that showed CD11b^pos/CD45^low expression (Sedgwick et al., 1991 (link)). Subsequently, for each brain region, the FACS sorted microglial cells were transferred into RNAse-free tubes and centrifuged for 5 min at 300x g (Hettich, Tuttlingen, Germany) at 4^∘C. The supernatant was aspirated, and cells were lysed with 500 μl Trizol reagent (Invitrogen, Carlsbad, CA, USA) for 5 min at RT. Lysates were stored at -80^∘C for subsequent RNA isolation, cDNA synthesis and qPCR analysis.