Anti cd29 pe

After pretreatment with TNF-α and IL-1β or the absence thereof, the hADSCs were harvested by digestion with 0.05% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Specialty Media, Millipore, Billerica, MA, USA) and washed with PBS twice. The cell pellets were resuspended in PBS and incubated with anti-CD29-PE, anti-CD44-FITC, anti-CD34-FITC, anti-CD45-FITC, anti-CD80-PE, anti-CD86-PE, anti-HLA-ABC-FITC, and anti-HLA-DR-PE (all from BioLegend, San Diego, CA, USA) for about 30 min in the dark. The cytometry data were processed and analyzed using FlowJo software.

In in vivo experiments, mice were sacrificed after tMCAO. The BALF was centrifuged at 500 g to collect cells. After being washed with PBS, cells were fixed and permeabilized (Invitrogen, Intracellular Fixation & Permeabilization Buffer Set), then stained with intracellular antibodies. The following antibodies were used: anti‐CD45‐BV421 (Biolegend, 103 134, clone: 30‐F11, 1:400), anti‐ F4/80‐AF488 (Biolegend, 123 120, clone: BM8, 1:400), anti‐LY6G‐APCCy7 (Biolegend, 108 424, clone: RB6‐8C5, 1:400), anti‐CD11c‐APC (Biolegend, 117 310, clone: N418, 1:400), anti‐CD3‐PECy7 (Biolegend, 100 220, clone: 17A2, 1:400), anti‐CD19‐PE (Biolegend, 152 408, clone: 1D3/CD19, 1:400), anti‐CD45‐PerCPCy5.5 (Biolegend, 368 504, clone: 2D1, 1:400), anti‐CD90‐FITC (Biolegend, 328 108, clone: 5E10, 1:400), anti‐CD34‐APC (Biolegend, 343 509, clone: 581, 1:400), and anti‐CD29‐PE (Biolegend, 303 004, clone: TS2/16, 1:400). Cell viability of BMDM was assessed using the Annexin V‐APC/PI apoptosis Detection Kit (KeyGEN, KGA1030‐100) according to manufacturer' s instructions.

Phenotype analysis was performed as described previously [77 (link)]. Passage 2 cADSC were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lake, NJ, USA; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), followed by blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated on ice for 20 min with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (BD Pharmingen, San Diego, CA, USA), anti-CD29-PE (BioLegend, San Diego, CA, USA), anti-CD34-PE (R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience, San Diego, CA, USA), and anti-CD90-PE (eBioscience) or their respective isotype controls. The cells were washed twice with FACS buffer and resuspended in 500 μL FACS buffer. Fluorescence was evaluated by flow cytometry in a CytoFLEX instrument (Beckman Coulter, Brea, CA, USA). The data were analyzed using CytExpert ver2.0 analysis software.

Passage 2 AT-MSCs were analyzed by flow cytometry. The cells were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated with the following fluorescein- (FITC-) or phycoerythrin- (PE-) conjugated antibodies: anti-CD14-FITC (BD PharMingen), anti-CD29-PE (BioLegend), anti-CD34-PE (R&D Systems), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience), and anti-CD90-PE (eBioscience) or their respective isotype controls listed in Table 1. The cells were washed twice with FACS buffer and resuspended in 500 μl FACS buffer. Fluorescence was evaluated by flow cytometry in a FACSCalibur instrument (BD Biosciences). Data were analyzed using WinMDI 2.9 analysis software.

The cell surface markers were analyzed with flow cytometry. The primary ADSCs at passage 3 (n = 3), and the ADSC-K4DT (n = 3) and ADSC-K4D (n = 3) cells were analyzed at low (approximately PDL 20), intermediate (approximately PDL 50), and high (approximately PDL 100) PDs. The cells were washed with FACS buffer (PBS containing 2% FBS), and the Fc receptors were blocked with canine Fc receptor binding inhibitor (Thermo Fisher Scientific). Then, the cells were incubated with the following phycoerythrin (PE)-conjugated antibodies: anti-CD29-PE (clone: TS2/16; BioLegend, San Diego, CA, USA), anti-CD34-PE (clone: 1H6; R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (clone: IM7; BioLegend), anti-CD45-PE (clone: YKIX716.13; eBioscience, San Diego, CA, USA), anti-CD90-PE (clone: YKIX337.217: eBioscience), and anti-HLA-DR-PE (clone: G46-6: BD Biosciences, Franklin Lake, NJ, USA). All the cells at each passage were examined in triplicate.

Muse‐AT cells were incubated at 4°C for 30 minutes in the presence of the following fluorochrome‐conjugated antibodies: anti‐SSEA3‐Alexa Fluor 647 (BD Biosciences), anti‐CD105‐APC, anti‐CD90‐FITC, anti‐CD73‐PE, anti‐CD29‐PE, anti‐CD34‐PE, anti‐CD73‐PE, anti‐CD45‐FITC, anti‐HLA‐ABC‐PE, anti‐HLA‐DR‐PE, and anti‐CD44‐PE (all from BioLegend). As a negative control, isotype‐matched irrelevant monoclonal antibodies were used. Analysis (10⁴ events per run) was performed using the FACSCantoII flow cytometer and DIVA6 software (BD Biosciences).

To examine the expression of ASC markers, cells (1 × 10⁵ cells) were incubated with anti-CD29-PE, anti-CD44-PE, anti-CD105-PE, anti-CD34-FITC, anti-CD45-PE, and anti-HLA-DR antibodies for 2 h at 4 °C (BioLegend, San Diego, CA, USA). Antibody-positive populations were quantitated using FACS-Calibur (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo (Tree Star, Ashland, OR, USA). Stain Index (SI) values for each (+) and (−) MSC biomarker were calculated using the following formula: $\mathrm{SI}=\frac{\mathrm{MFI}1-\mathrm{MFI}2}{2\times \mathrm{SD}}$ ; MFI1, geometric mean fluorescence of control stain; MFI2, geometric mean fluorescence of test stain; SD, standard deviation of geometric mean fluorescence.