Apc anti mouse cd31

MSCs were detected the expression of specific surface markers, including PE anti-mouse Sca-1, PE anti-mouse CD90.2, PE anti-mouse CD29, PE anti-mouse CD44, PE anti-mouse CD45, APC anti-mouse CD31, APC anti-mouse CD34, and APC anti-mouse CD117 (Biolegend, San Diego, CA).
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.

SVFs were isolated as described previously.^[58] Briefly, inguinal fat from Mettl3 FKO or WT mice was minced and digested with 2 mg mL⁻¹ collagenase type II (Sigma, C6885) in PBS supplemented with 1% Hepes at 37 °C for 20–30 min, followed by quenching with complete medium. Cell suspensions were centrifuged, washed, and filtered through a 40‐µm strainer. For fluorescence activated cell sorting (FACS) analysis, SVFs were resuspended in red blood cell lysis buffer (Beyotime, C3702) for 15 min at room temperature and then further incubated with PE anti‐mouse Pdgfrα (BioLegend, 135 905), APC anti‐mouse CD31 (BioLegend, 102 509), APC anti‐mouse CD45 (BioLegend, 103 116), and subjected to FACS.

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).

Mammary glands from 2-week-old (precisely 14 days old), 5-week-old (between 4.7–5 weeks and designated 5 weeks herein) and 10-week-old female mice were collected and single-cell suspensions were prepared and stained^{45 (link)}. The following antibodies were used: FITC anti-mouse CD29 (rat, clone HMβ1-1, BioLegend Cat#102206, 1/200 dilution), Pacific Blue anti-mouse CD24 (Armenian Hamster, clone M1/69, BioLegend Cat#101820, 1/200), APC anti-mouse CD31 (rat, clone 390, BioLegend Cat#102410, 1/40 dilution), APC anti-mouse CD45 (rat, clone 30-F-11, BioLegend Cat#103112, 1/100 dilution), APC anti-mouse TER-119/erythroid cell (rat, clone TER-119, Biolegend Cat#116212, 1/100 dilution), PE anti-mouse CD55 (Armenian hamster, clone RIKO-3, BioLegend #131803, 1/50 dilution), and biotin anti-mouse CD14 (rat, clone Sa2–8, eBioscience #13-0141-85, 1/100 dilution). To exclude dead cells, cells were re-suspended in 0.5 μg/ml propidium iodide prior to analysis. FACS analysis and cell sorting were performed on a FACS Aria (Becton Dickinson). The Lin^– population was defined as Ter119^–CD31⁻ CD45^–. FACS data were analyzed using FlowJo software (v 10.1r7, Tree Star).

For FACS analysis in vitro cultured cells and tissues were prepared as previously described (12 (link)). In the case of the lung, tissue was digested in collagenase IV-containing Grey's Balanced Salt Solution (GBSS, Sigma-Aldrich). Cell suspensions were stained with anti-mouse CD31-APC, anti-mouse CD45-PerCP and podoplanin-PE (all from Biolegend) to identify LECs (CD45⁻CD31⁺podo⁺) and BECs (CD45⁻CD31⁺podo⁻), respectively. ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen). Some FACS experiments were performed using I/F8-Fc (2 μg/ml) or KSF-Fc (2 μg/ml) followed by incubation with anti-mouseAlexaFluor488 or -PE-labeled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Data were acquired on a BD FACSCanto (BD Bioscience, Franklin Lake, NJ, USA) using FACSDiva software (BD Bioscience) and analyzed with FlowJo software 8.7.1. (Treestar, Ashland, TN, USA).