Pe cy 5 anti human cd45

Leukocytes from mice blood, spleen, and bone marrow (BM) were quantified using flow cytometry. Briefly, 100 μL peripheral blood was collected in EDTA anticoagulant tubes. Single-cell suspensions of the spleen were obtained through mechanical disaggregation. Single-cell suspensions of the BM were flushed from femurs and tibias. These single-cell suspensions were then filtered through 70 μm cell strainers (15-1070, Biologix, West Hollywood, CA, USA). The erythrocytes were lysed using RBC lysis buffer (R1010, Solarbio, Beijing, China). After 2 washes with staining buffer, cells were preincubated with Human TruStain FcX™ (422302, Biolegend, San Diego, CA, USA) for 10 min at room temperature, followed by staining with fluorochrome-labeled antibodies (1:50) in the dark at 4 °C for 45 min, and detection with an LSRfortessa flow cytometer (BD Biosciences, San Jose, CA, USA). The following fluorochrome-conjugated antibodies were used: PE-Cy™5 anti-human CD45 (555490, BD Biosciences), PE anti-mouse CD45 (561087, BD Biosciences), APC R700 anti-human CD3 (659119 BD Biosciences), APC anti-human CD4 (565994, BD Biosciences), FITC anti-human CD8 (555634, BD Biosciences), BV421 anti-human CD19 (562440, BD Biosciences). The data were analyzed employing FlowJo software.

Muscle stem/progenitor cells were isolated as described previously^{31 (link),53 (link)}. Briefly, muscle tissues were sliced to 1 mm³ pieces, digested by collagenase II (Worthington biochemical, 700-800 U/mL, cat#LS004177) for an hour and subsequently digested by mixtures of collagenase II and dispase (Life Technologies,11 U/mL, cat#17105-041) for 30 min. The digested mixture was passed 10 times through a 20-gauge needle and filtered through a 40-µm cell strainer (BD Falcon, cat#352340). The erythrocytes were removed by red blood cell lysis (Thermo Fisher Scientific, cat#00-433-57). The human cell suspension was stained with PE-Cy5 anti-human CD45 (BD Pharmingen, cat#555484, 1:25), Percp-Cy5.5 anti-human CD31 (BioLegend, cat#303132, 1:100), AF-488 anti-human CD29 (BioLegend, cat#303016, 1:100) and BV421 anti-human CD56 (BD, cat#562751, 1:100) for 45 min at 4 °C. The mouse cell suspension was stained with a cocktail of APC anti-mouse CD31 (BioLegend, cat#102510, 1:100), APC anti-mouse CD45 (BioLegend, cat#103112, 1:100), FITC anti-mouse Sca1 (BioLegend, cat#108106, 1:100) and Biotin anti-mouse VCAM1 (BioLegend, cat#105703, 1:100) for 45 min at 4 °C. All cell suspensions were washed with PBS and stained with PE/Cy7 Streptavidin (BioLegend, cat#405206, 1:100) for 15 min and sorted by FACS using Aria III or Influx (BD Biosciences).