Pe cy5 anti cd4

The procedure and protocol of flow cytometry for identification and quantification of circulating and splenic immune cells were based on our previous report [44 (link)]. Briefly, the peripheral blood mononuclear cells (PBMCs) and splenocytes were obtained from the tail vein using a 27# needle. PBMCs and splenocytes (1.0 × 10⁶ cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience), and PE-Cy™5 anti-CD4 (BD bioscience). The numbers of CD3⁺CD4⁺ helper T cells and CD3⁺CD8⁺ cytotoxic T cells were analyzed using flow cytometry (FC500, Beckman Coulter).

Freshly isolated splenocytes were stained with PE-Cy5 anti-CD4 and PE-anti-CD25 or isotypes rat IgG2a,κ and rat IgM,κ, respectively, for 10 min at 4 °C (BD). Cells were then fixed and permeabilized using the Mouse FoxP3 Buffer Set and then incubated with Alexa Fluor 488 anti-FoxP3 or isotype rat IgG2baccording to manufacturer’s instructions (BD). Flow cytometry was performed immediately or within 24 h of permeabilization using a FACScaliber machine. 1 × 105 gated events were recorded. Results were analyzed with FCS Express software.

The following antibodies from BD Biosciences were used for flow cytometry: biotinylated antibodies to CD4 (553728), CD8 (553029), IgM (553436), CD11b (553309), CD138 (553713) and GR-1 (553125); PE-conjugated antibodies to B220 (553090), CD5 (553023), CD11b (557397), IgD (558597), CD138 (553714); APC-conjugated antibodies to CD21 (558658), IgM (550676), IgD (560868), CD11b (553312), CD138 (558626) and GR-1 (553129); FITC-conjugated antibodies to GL7 (553666), IgD (553439); PE-Cy7 conjugated anti-CD11b (552820); Pacific Blue anti-CD3e (558214); PE-Cy5-anti-CD4 (553050) and APC-Cy7-anti-CD8a (557654). From Biolegend: APC-anti-mouse IgG1 (406610), PerCPCy5.5-anti-mouse IgG1(406612), Pacific Blue anti-CD38 (102720), FITC-anti-mouse IgG (405305), APC-anti-mouse IgG (405308), APC-Cy7-anti-mouse IgG (405316). From eBioscience: PerCP-Cy5.5-anti-B220 (45-0452-82), PE-anti-IgM (12-5890-83), PE-anti-CD23 (12-0232-82), Biotin-anti-mouse-IgD (13-5993-85), Streptavidin-PE (12-4317-87), PE-Cy7-streptavidin (25-4317-82). From the Jackson Immunoresearch Laboratories: normal rabbit IgG (015-000-002) or mouse IgG (011-000-002). From Abgent: anti-LC3 (AP1802a) for immunocytochemistry. From Invitrogen: Anti-CoxIV (459600) for immunocytochemistry. From Southern Biotechnology, HRP conjugated anti-mouse IgG or IgM.

After 2 days co-culture of hNSCs and PBMCs, we aspirated suspensions contained PBMCs using plastic Pasteur pipette and washed cells for use. For cell surface marker staining, the following fluorochrome-conjugated anti-human antibodies were used (all from BD Biosciences): polyethylene (PE) anti-TCRγδ (B1), FITC anti-CD3 (UCHT1), PE-CY5 anti-CD4 (RPA-T4), PE anti-CD8 (RPA-T8). Cells were incubated with antibodies and washed once with PBS before analysis on a BD FACS Calibur flow cytometer. For Treg cell detection, FITC anti-CD4 (RPA-T4), allophycocyanin (APC) anti-CD25 (M-A251) and PE anti-FOXP3 (259D/C7) were used according to manufacturer’s instructions. For cell sorting, stained cells were sorted on a FACS machine (BD Calibur, USA) and the results were analyzed using FlowJo7.6.1 software.

CD4⁺ CD25⁺ Treg cells and CD4⁺ CD25⁻ Teff cells were isolated first using EasySep Mouse CD4⁺ T-Cell Isolation Kit (Stem Cell), then enriched cells were labeled with PE-Cy5-anti-CD4 and PE-anti-CD25 (BD) and flow sorted on a FACS Vantage (BD). Dendritic cells (DCs) were generated from bone marrow (BM) from either syngeneic (C57BL/6) or allogeneic (BALB/C) mouse strain as previously described (Yan et al., 2009 (link)). CD11c⁺ DCs from both conditions were purified with mouse CD11c MicroBeads Kit as per manufacturer instructions (Miltenyi biotec). Of 4 × 10⁴ Teff cells from 3–4 months old wild-type C57BL/6 were incubated in the indicated ratios in the T-cell media with Tregs from young and old mice in the presence of 5 × 103 CD11c⁺ DCs for ~5 days in round bottom 96-well plates. Media were supplemented with 1 μg mL⁻¹ anti-CD3e (BD) when coculturing syngeneic BM-derived and splenic DCs. Anti-IL10 neutralization antibodies or N-acetyl cysteine (NAC), when used, was administered as a bolus to a final concentration of 10 μg mL⁻¹ or 2 mm, respectively, at the start of coculture. Incorporation of 3H-thymidine (1 μCi/well, Perkin Elmer) by proliferating cells was measured during the last 18 h of culture.