Anti cd45 apc

Single-cell suspension of splenic or lymph node cells were incubated with 10% 2.4G2 culture supernatants to block Fc receptors and then stained with indicated antibodies in MACS buffer (PBS supplemented with 1% FBS and 5mM EDTA). Staining reagents include: APC-Cy7 anti-CD19 (1D3), APC-Cy7 anti-B220 (RA3-6B2), AF700 anti-CD4 (RM 4-5), APC anti-CD4 (GK1.5), FITC anti-CD4 (gk1.5), AF700 anti-CD44 (IM7), APC anti-CD45.1 (A20), eF450 anti-CD45.2 (104), PE-Cy7 anti-CD95 (Jo2), eFlour450 anti-GL7 (GL7), PE-Cy7 anti-PD-1 (RMP1-30), PE anti-IgM^a (DS-1), FITC anti-IgD (11-26c) from eBioscience; biotinylated anti-CXCR5 (2G8), PE anti-CD40L (MR1), streptavidin PE (Cat 554061) and streptavidin APC (Cat 554067) from BD Biosciences. Dead-cell exclusion was based on 7-AAD staining (Biotium), and non-singlet events were excluded with FSC-H/FSC-W and SSC-H/SSC-W. Isotype-matched non-specific antibodies were purchased from the corresponding companies. Cells were stained with primary antibodies for 60-90 min, washed, and then with secondary reagents for 30 min on ice. For intracellular staining, cells were stained using Cytoperm/Cytofix kit (BD Biosciences) according to the manufacturer’s protocol. All flow-cytometry data were collected on an LSR-II or Aria III (BD).

The following antibodies were used for FACS: FITC-anti-CD11b (11–0112), APC-anti-CD11b (17–0112), PE-anti-F4/80 (12–4801), FITC-anti-F4/80 (11–4801), APC-anti-CD45 (17–0451), PerCp-Cy5.5-anti-CD45 (45–0451), FITC-anti-CD24 (11–0242), APC-anti-CD24 (17–0242), PerCP-Cy5.5-anti-NK1.1 (45–5941), PE-anti-CD4 (12–0041), FITC-CD3e (11–0031), PerCP-Cy5.5-Gr1 (45–5931), and PE-anti-Ly6C (12–5932) obtained from eBioScience, and PE Annexin V Apoptosis Detection Kit I (559763) from BD Pharmingen. Cells were blocked for 15 min with Fc blocking reagent on ice prior to labeled with fluorescent-conjugated antibodies diluted in FACS buffer. Cells for phenotypic analyses were stained using the indicated fluorescent-conjugated antibody for 30 min on ice. Appropriate isotype controls were used in all case. Flow cytometry was performed using a FACSCalibur (BD Bioscience, San Jose, CA) and the data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Whole blood was used for membrane staining and one-step density gradient centrifugation (Lymphoprep: d = 1.077 g/mL; Nycomed Pharma AS, Oslo, Norway) was used for intracellular FoxP3 labeling in isolated mononuclear cell fractions. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) with CellQuest Pro software (Becton Dickinson) to acquire cells. The following monoclonal antibodies (Mo-Abs) were used for cell surface and intracellular staining after permeabilization: PerCP-anti-CD4, FITC-conjugated anti-CD25, APC-anti-CD45, and PE-conjugated anti-FOXP3 (eBioscience, San Diego, CA, USA).
Phenotypic analyses were performed using WinMDI software. Generally, 20,000 cells were acquired. Lymphocytes were analyzed in the gated population, which lacked cells with granulocyte or monocyte characteristics (CD45high, SSC low). In 16 patients, the proportion of CD4+CD25high lymphocytes correlated with the proportion of FoxP3 + CD4+ lymphocytes (R = 0.551, p = 0.028).

APC-anti-CD45, PE-anti-CD105 and PE-cy7-anti-Sca1 antibodies (Abs) are purchased from eBioscience. Callus and BM cells are stained with various fluorescein-labeled Abs and subjected to flow cytometric analysis using a Becton-Dickinson FACSCanto II Cytometer, according to the manufacturer’s instructions. Results are analyzed by Flowjo7 data analysis software (FLOWJO, LLC Ashland, OR). For cell sorting, BM cells from Nestin-GFP mice are sorted using a Becton-Dickinson FACSAria III sorter, according to the manufacture’s instructions [29 ].