Pe rat anti mouse cd45

Brain samples were collected in sham or MCAO mice, digested with collagenase IV and DNase, and filtered with sterile cell strainers (Biologix, 70 µm). Single-cell suspensions were prepared after the removal of red blood cells by ammonium-chloride-potassium (ACK) lysis buffer and isolated leukocytes in brains by 30%, 37%, and 70% Percoll (GE Healthcare) density gradient centrifugation, respectively. Cell numbers were counted by a Coulter counter (Thermo Fisher). Cells were washed with buffer (PBS with 0.5% bovine serum albumin and 0.02% sodium azide) three times and subsequently stained with fluorochrome-conjugated monoclonal antibodies: PE Rat anti-Mouse CD45 (553081, BD Biosciences), FITC anti-mouse F4/80 antibody (123108, Biolegend), PerCP-Cyanine5.5 CD11b antibody (45-0112-82, Invitrogen), and APC anti-mouse I-A/I-E antibody (107614, Biolegend). Samples were analyzed using Flow Cytometry (CytoFLEX S). Subsequent analysis was performed with FlowJo software (Tree Star Inc., San Carlos, CA, USA). Total microglia were identified as CD45⁺F4/80⁺CD11b⁺, M1-like microglia were identified as CD45 + F4/80 + CD11b + MHCII + .

BM cells from normal, 5-FU-treated, and 5-FU + FSH-treated mice were used for flow cytometry to enumerate Sca-1⁺/Lin^–/CD45^– VSELs and Sca-1⁺/Lin^–/CD45⁺ HSCs using the gating strategy described by Kucia et al. [6 (link)]. A single-cell suspension was prepared and stained with FITC-conjugated rat anti-mouse SCA-1 (BD Biosciences, San Jose, CA, USA), PE rat anti-mouse CD45 (BD Biosciences), and APC mouse Lineage antibody cocktail (BD Pharmingen, San Diego, CA, USA) for 60 min on ice. After washing, the stained cells were run on FACS Aria (BD Biosciences). At least 10⁵ events were acquired and results were analyzed by using BD FACS Diva software (BD Biosciences).

The cells were subjected to staining using the 7-AAD Viability Staining Solution (Invitrogen, 00-6993-50) and lineage markers, rat anti-mouse CD11b-PE (BD Pharmingen, 557397), rat anti-mouse CD31-PE (BD Pharmingen, 553373), rat anti-mouse CD45-PE (BD Pharmingen, 553081), biotin rat anti-mouse TER-119 (BD Pharmingen, 553672), and streptavidin-PE (BD Pharmingen, 554061). The cells were then allowed to pass through a 35-μm cell strainer (Corning, 352235). Single cells were sorted using SH800 (SONY, Tokyo, Japan) in 2% bovine serum albumin/PBS. Sorted cells were subjected to processing using the Chromium Controller (10x Genomics, Pleasanton, CA, USA), Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000128), and MGIEasy Universal Library Conversion Kit (App-A) (MGI, Shenzhen, China) following the manufacturer’s instructions. The library was sequenced using DNBSEQ-G400 (MGI).

For isolation of SCs by FACS sorting, skeletal muscles from hind limbs were prepared similarly as for flow cytometry analysis, resuspended in PBS + 2% FBS, and then incubated with the following antibodies for 30 min on ice: rat anti-mouse α7integrin-APC (1:15, 334,908, R&D Systems), rat anti-mouse CD34-FITC (1:30, RAM34, eBioscience), rat anti-mouse CD45-PE (1:30, 30-F11, BD Biosciences), rat anti-mouse CD31-PE (1:30, MEC13.3, BD Biosciences), and rat anti-mouse Sca-1-PE-Cy7 (1:30, D7, eBioscience). After incubation, cells were washed, filtered through a 40-μm cell strainer, and resuspended in PBS + 2% FBS with Hoechst 33342 (10 μg/ml) and 7-AAD (1:40, BD Biosciences). Cells were sorted with MoFlo XDP (Beckman Coulter) cell sorter.