Cd45 efluor450

Adipose tissue fractionation and flow cytometric analysis were performed to identify adipose tissue macrophage (Singer et al., 2014 (link)). Briefly, whole adipose tissue was minced and digested with type II collagenase (1 mg/mL, Sigma, #C6885) for 15 to 30 min at 37°C on a rocker. Samples were filtered and centrifuged, and red blood cell lysis was conducted on SVF. SVF single cell suspensions were then incubated with the antibodies. The following antibodies were used: CD45 eFluor450 (Clone 30‐F11, Invitrogen), CD64 PE (Clone X54‐5/7.1, BD Pharmingen), CD11c APC eFluor780 (Clone N418, Invitrogen), CD45 FITC (Clone 30‐F11, Biolegend), F4/80 PE (Clone BM8, Biolegend), and CD11b BV421 (Clone M1/70, Biolegend). Flow cytometry was performed using a BD LSRFortess flow cytometer and analyzed with FlowJo V10.7 software. Macrophages were defined as CD45⁺CD64⁺ or CD45⁺CD11b⁺F4/80⁺, and infiltrated macrophages were defined as CD45⁺CD64⁺CD11c⁺(Singer et al., 2015 (link)).

P3 JBMMSCs were digested using trypsin before their resuspension in α-MEM with 10% FBS. Phosphate-buffered saline (PBS) was employed for washing the cells twice. Following this, the cells were incubated for 30 min with antibodies against CD45-eFluor 450, anti-CD34-PE, anti-CD44-FITC, anti-CD146-PE, anti-CD90-APC (Invitrogen, USA), and anti-CD31-FITC (Bioss, China). The cell suspension was then subjected to centrifugation for 5 min at 1000 rpm. Lastly, the cell suspension was moved to a fresh detection tube, and cell surface antigen was detected making use of flow cytometry (BD Biosciences, USA).

Perfused mouse brains were collected in RPMI medium, ground gently to disperse tissue, and spun in 90% Percoll (P1644; Sigma) with a 63% Percoll underlay to isolate leukocytes at the interface. Leukocytes were resuspended in fluorescence-activated cell sorting (FACS) buffer (1× PBS, 1% bovine serum albumin) and stained with the appropriate fluorescently labeled antibodies. Labeled cells were fixed for 20 min in 4% paraformaldehyde before analysis on an LSRFortessa instrument (BD Biosciences). Antibodies used in this study (eBiosciences) were the following: CD45-efluor450 (catalog no. 48-0451-82), CD4-allophycocyanin (APC) (17-0041-82), CD8-fluorescein isothiocyanate (FITC) (11-0081-82), F4/80-FITC (11-4801-82), Ly6G-FITC (11-5931-82), and Ly6C-APC (17-5932-82).

Mononuclear cells were isolated from brain and spinal cord of CX3CR1-WT, CX3CR1-KO, and hCX3CR1^I249/M280 mice by homogenization of CNS tissues as previously described (Pino and Cardona, 2011 (link)). Cellular suspensions were blocked using anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmingen) followed by incubation for 30 min with a mix of fluorochrome-conjugated anti-mouse antibodies as follows. Antibody cocktail for mononuclear myeloid cells and activation markers: CD45 APC-Cy7 (clone 30-F11, BioLegend), CD11b PE-CF594 (clone M1/70, BD Biosciences), CD11c PE-Cy7 (clone N418, eBioscience), I-A/I-E BV421 (MHC-II clone M5/114.15.2, BD Biosciences), CD86 PerCP/Cy5.5 (clone GL-1, BioLegend), Ly6C AF647 (clone ER-MP20, Serotec), Ly6G PE (clone 1A8, BD Pharmingen). T cells were characterized using the following antibody mix: CD45 eFluor 450 (clone 30-F11, eBioscience), CD3e PE-Cy7 (clone 145-2C11, BioLegend, CD8a APC (clone 53-6.7, eBioscience), CD4 APC-Cy7 (clone RM4-5, BioLegend), CD44 PerCP (clone IM7, eBioscience). Samples were acquired on an LSRII (BD Biosciences) at the Cell Analysis Core, UTSA. Data analysis was performed using FlowJo v10. For cell sorting, brain cell suspensions (pooled 3 mice per sample) were stained with antibodies against CD45 and CD11b and after gating for single cells the CD45^LoCD11b⁺ population was separated in a FACS ARIA-II (BD Biosciences).