Pe conjugated anti cd4

C11 and A10.6 cells were incubated with various concentrations of drugs for different times. The cells were then washed and harvested in cold PBS, and the GFP expression was measured using a BD FACScan Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The cells were analyzed using Cell Quest software Version 6.1 (BD Biosciences). Live cells were gated, and two parameter analyses were used to differentiate GFP-associated fluorescence from background fluorescence. A total of 10,000 gated events were collected per sample. The data represent the percentage of GFP-expressing cells in all gated events.
The PBMCs were treated with Scopoletin or Prostratin. Next, 0.5 × 10⁶ cells were collected and stained with FITC-conjugated anti-CD25 (BD Biosciences, 560990) and PE-conjugated anti-CD69 antibodies (BD Biosciences, 560968) to analyze T cell activation. PE-conjugated Anti-CD4 (BD Biosciences, 561843), FITC-conjugated anti-CCR5 (BD Biosciences, 561747), and FITC-conjugated anti-CXCR4 antibodies (MBL D123-4) were also used to stain the treated PBMCs. The treated PBMCs were washed with PBS and incubated for 30 min on ice with 2 µL of antibodies in 100 µL of PBS. Subsequently, the cells were washed three times and harvested in PBS. A total of 10,000 gated events were collected per sample.

The expression of T cell and B cell lineage receptors was assessed by phenotyping using the following monoclonal antibodies (all purchased from BD, CA): antihuman FITC-conjugated antiCD3, PE-conjugated antiCD4, PerCP-conjugated antiCD8, APC-conjugated antiCD19, PC7-conjugated antiCD38, and the corresponding isotype controls. According to the manufacturer’s instructions, peripheral blood mononuclear cells were stained with these antibodies at concentrations titrated for optimal staining. The multicolor staining of monoclonal antibodies was carried out in the following panel: CD3/CD4/CD8/CD19/CD38.

For intracellular cytokine staining, T cells were treated for 2 h with brefeldin-A (Golgi plug; BD Pharmingen) at a concentration of 2 μg/mL, then harvested and washed in phosphate-buffered saline, containing 1% FBS and 0.1% NaN₃ (staining buffer). They were then stained extracellularly using PerCP-conjugated anti-CD3, PE-conjugated anti-CD4, APC-Cy7-conjugated anti-CD8 monoclonal antibodies or the appropriate isotype controls for background determination, all purchased from BD Pharmingen (San Diego, CA, USA). Then, the cells were fixed and permeabilized using Cytofix/Cytoperm™ (BD Pharmingen), according to the manufacturer’s instructions, and stained with PeCy7-conjugated anti-human IFN-γ and APC-conjugated TNF-α (BD Pharmingen). Stained cells were then analyzed with flow cytometry using the Beckman Coulter Gallios equipped with Kaluza Software (Beckman Coulter, Brea, CA, USA).

After lung cell purification, intracytoplasmic cytokine staining was performed as previously described [26 (link), 27 (link)]. The cells were stained for cell surface markers with PE-conjugated anti-CD4 (BD Biosciences, Franklin Lakes, NJ, USA), FITC-conjugated anti-CD8 (BD Biosciences), or APC-conjugated anti-CD3 (BD Biosciences) and then analyzed using a MACS Quant flow cytometer (Miltenyi Biotec, Auburn, CA, USA) with FlowJo software (TreeStar, Ashland, OR, USA). The numbers of cytokine-producing CD4⁺ or CD8⁺ T cells per lung were calculated from the percentages of cytokine-producing cells and the numbers of CD4⁺ or CD8⁺ T cells isolated from the lung. The cells were also stained for cell surface markers with APC-conjugated anti-CD11b (BD Biosciences) and FITC-conjugated anti-CD11c (BD Biosciences).

For primary NK cells, single hepatic lymphocytes from 6- to 8-week-old C57BL/6 mice were labeled with PE-Cy7-conjugated anti-CD3, PE-conjugated anti-CD4, APC-conjugated anti-NK1.1 and APC-Cy7-conjugated anti-CD19 (BD PharMingen, United States). NK cells were sorted as cells that express NK1.1⁺CD3^- using a FACS Aria I flow cytometer (BD Biosciences, United States) and the purity was higher than >95%. After purification, NK cells were cultured in a 96 round bottom wells (Costar, Corning Incorporated, Corning, NY, United States) in complete RPMI 1640 medium supplemented with 10% (v/v) fetal serum (Gibco, United States), 200 mg/ml glutamine, 50 μg/ml gentamycin, 100 units/ml penicillin, 100 μg/ml streptomycin (Sangon Biotech, Shanghai, China) and interleukin-2 (IL-2, 10 U/ml, BD PharMingen, United States).

In immunization-based EAMG models, antigens are injected subcutaneously to body parts that are in close proximity with the lymph nodes. As a result, immunopathogenic processes leading to antibody formation and muscle weakness predominantly occur in the local draining lymph nodes. Therefore, in our study, all immunopathological studies were conducted using lymph node cells. For this purpose, inguinal, popliteal, and axillary lymph node cells were collected at the termination of the experiment. Single-cell suspensions of lymph node cells were incubated for 30 min with one of the following anti-mouse antibodies: PE-conjugated anti-CD4, anti-CD19, anti-CD11b, and anti-CD3 and FITC-conjugated anti-CD8 (all from BD PharMingen). PE- or FITC-conjugated isotypes were used for controls. Cells were washed twice and then were fixed with 2 % paraformaldehyde and analyzed by flow cytometry (BD Biosciences).