Streptavidin pe cy7

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).

Mouse peripheral blood was processed to remove RBC via lysis prior to Fc blocking and then stained with antibodies against surface markers including biotin-CD93 (Invitrogen, cat # 13-5892-85), BV605-CD19 (BD, cat # 115539), APC Cy7-CD3 (BioLegend, cat # 100222), Alexa Fluor 647-B220 (BioLegend, cat # 103226), and PE Cy7-streptavidin (BioLegend, cat # 405206). The cells were washed with complete RPMI-1640 pre-warmed to 37°C and stained with Indo-1 AM (Invitrogen, cat # I1223) diluted to 2 μM in warm RPMI-1640 and incubated at 37°C for 30 min before washed and stained with 7-AAD (Invitrogen, cat # A1310).
For acquisition, data scales of the detectors for the BUV395 (379/28) and BUV496 (515/30 450LP) filters were set to linear. Samples were kept at 37°C prior to and during acquisition. Baseline was recorded for 30 sec, followed by the addition of anti-mouse IgM (Jackson ImmunoResearch; cat # 115-006-075) to a final concentration of 10 μg/mL, immediately mixed before resuming acquisition for further 3.5 mins to measure the calcium flux following BCR ligation. At 4 mins post-acquisition, ionomycin (Invitrogen, cat # I24222) was added to the sample tube to a final concentration of 1 μg/mL, immediately mixed before before resuming acquisition for another 4 mins to measure maximal Ca²⁺ flux output.

CD3-FITC clone 145–2C11, CD4-PE-Cy7 clone RM4–5, CD4-PE clone RM4–4, CD11a-FITC clone M17/4, CD49d-PE clone R1–2, CXCR5-biotin clone L138D7, PE-Cy7 streptavidin, CD44-FITC and CD44-APC-Cy7 clone IM7, PD-1 APC clone 29F.1A12, GL7-PacBlue and GL7-PE clone GL7, CD19-PerCP-Cy5.5 clone 6B2, CD138-BV421 clone 281–2, IgD-APC-Cy7 clone 11–26c2a, CD95-PE clone SA367H8, CD73-BV605 clone TY/11.8, CD71-BV421 Clone RI7217, and CD80-PE clone 90, were purchased from Biolegend (San Diego, CA). BcL6-PE-CF594 clone K112–91 was purchased from BD Biosciences, San Jose, CA)