Anti cd19 apc

The surface markers of PDLSCs were examined using a flow cytometer (Calibur, BD Biosciences). 1 × 10⁶ PDLSCs were incubated with following luorochrome-conjugated antibodies: anti-CD44-APC (Cat. MA1-10,226, Invitrogen), anti-CD105-PE (Cat. 12–1057–42, Invitrogen), anti-CD90-PE (Cat. 12–0909–42, Invitrogen), anti-CD45-FITC (Cat. 14–9457–95, Invitrogen), anti-CD19-APC (Cat. 17–0199–42, Invitrogen) and anti-CD14-PerCP (Cat. 450,149–42, Invitrogen) for 1 h at 4°C in the dark and were then analyzed after washing in PBS.
The percentage of cell apoptosis was determined by FACS. Apoptotic PDLSCs were detected according to the instructions of the AnnexinV-APC/7-AAD apoptosis detection kit (BestBio, Shanghan, China). Briefly, PDLSCs were trypsinized and the resuspended. PDLSCs were washed with PBS and stained with annexin V-APC and 7-AAD. The cells were then analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, United States).

Eight cell surface markers (CD105, CD73, CD90, CD45, CD34, CD14, CD19, and HLA-DR) were evaluated to identify MSCs according to ISCT criteria (Dominici et al., 2006 (link)). MSCs were counted to ensure each cell suspension had more than 1 × 10⁶ cells. The cells were incubated with human anti-CD105 (PE), anti-CD73 (FITC), anti-CD90 (PE), anti-CD45 (PE), anti-CD34 (PE), anti-CD14 (FITC), anti-CD19 (APC), and anti-HLA-DR (FITC) (all from eBioscience, San Diego, CA, USA), respectively for 30 min at room temperature. The cells were then washed and suspended for flow cytometry analysis.

Cell surface markers were stained with these specific antibodies: anti-CD19-PE, anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC, 7-AAD, and anti-annexin-V-FITC (eBioscience); anti-CD19-APC, anti-B220-Per-cy5.5, anti-IgM-FITC, anti-AA4.1-PE, anti-CD23-eFluor647, and anti-CD8-APC (Biolegend).
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO₂. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.

Cells were seeded into 12-well plates at 0.8 × 106 cells per mL per well. Treated cells were harvested and washed twice with PBS-EDTA. Cell death was determined using annexin-V-FITC and PI (propidium iodide) staining according to the manufacturer’s guidelines (BD Pharmingen, Erembodegem, Belgium). To determine cell death in B lymphocytes, PBMCs were immunostained with anti-CD45 (e-Fluor 780) and anti-CD19 (APC) antibodies (eBioscience, San Diego, CA, USA) prior to annexin-V-FITC/PI staining. Stained cells were analyzed using flow cytometry (NAVIOS-Beckman Coulter, Indianapolis, Indiana), and the generated data were analyzed using KALUZA software (Beckman Coulter).

5-6 C57BL/6 male mice aged 16 weeks received 0.5 mg of depleting anti-CSF1R mAb (BioXCell, clone AFS98) or isotype control (BioXCell, Rat IgG2a) intraperitoneally on day −7, −4 and −2 prior to euthanasia per experiment (n = 2 independent replicates). Following terminal procedure, kidneys, liver and prostate from each mouse were harvested and divided for either flow cytometry, microscopy or zinc quantification. Tissues for flow cytometry was minced finely and digested in RPMI containing 0.1 mg/mL DNase I, 32.5 mg/mL Liberase TM and 10 mM HEPES for 25 min at room temperature. Organs were then mechanically dissociated through a 70 μm cell strainer, washed in PBS and red blood cell lysis performed. Single cell suspensions were blocked for 30 min with 50 μL normal mouse serum in PBS 2% FBS on ice then stained with live/dead fixable aqua (Invitrogen), anti-CD45 APC-eFluor780 (30-F11, eBioscience), anti-I-A/I-E Pacific Blue (M5/114.15.2, Biolegend), anti-CD11b PerCP-Cy5.5 (M1/70, Invitrogen), anti-F4/80 PE/Cyanine7 (BM8, Invitrogen), anti-CD3e APC (145-2C11, Invitrogen), anti-CD19 APC (1D3, eBioscience) and anti-Ly-6G/Ly-6C APC (Rb6-8C5, Biolegend).

Small intestine and colon tissues were incubated at 37 °C in PBS containing 2% FBS and 5 mm EDTA for 25 min. The remaining tissue was cut into small pieces and digested in PBS containing 2% FBS, Collagenase IV (0.5 mg mL⁻¹; Thermo), and DNase I (10 U mL⁻¹; Sigma‐Aldrich) and then incubated at 37 °C for 45 min. Single cell suspensions were stained with anti‐CD45‐Alexa Flour 700 (eBioscience, 30‐F11, 56‐0451‐82, 1:400), anti‐CD4‐APC‐Cy7 (Biolegend, GK1.5, 100 414, 1:400), anti‐CD8a‐ PE (eBioscience, 53–6.7, 12‐0081‐83, 1:400), anti‐NK1.1‐PE‐Cy7 (eBioscience, PK136, 25‐5941‐82, 1:400), anti‐CD19‐APC (eBioscience, 1D3, 17‐0193‐80, 1:400), anti‐CD11b‐FITC (Biolegend, 101 206, M1/70, 1:400), anti‐CD11c‐PE (eBioscience, N418, 12‐0114‐82, 1:400), and FVD eFlour 506 (eBioscience,1:1000) for FACS analysis (Thermo). All flow cytometry analyses were performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo 10 software.