Fitc conjugated anti cd8

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).

WHV-specific CD8⁺ and CD4⁺ T cells were detected by established methods described in previous publications [10 (link), 20 (link)]. Splenocytes in 96-well U-bottom plates were re-stimulated with individual peptides at a concentration of 2 μg/ml in the presence of brefeldin A (eBioscience, San Diego, CA) at 4 μg/ml for 5 h. All antibodies were purchased from Biolegend (San Diego, CA). Splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 and phycoerythrin (PE)-conjugated anti-CD4, while dead cells were excluded by 7-AAD staining (BD pharmingen, NJ). For intracellular IFN-γ staining, cells were permeabilized by Fixation & Permeabilization kit (eBioscience) and stained with allophycocyanin (APC)-conjugated anti-IFN-γ (Biolegend). Data were analyzed using FACS Calibur (BD Biosciences, San Jose, CA) and FlowJo software (Treestar, Ashland, OR).

Spleen cells from the mice about 7 days after the third immunization were separated by lymphocyte separation medium and resuspended. OVA-specific CTLs were generated by stimulating spleen lymphocytes with 50 μg/mL of OVA protein for 3 days. The cells were then stained by FITC-conjugated anti-CD8 (BD Biosciences) and PE-conjugated H-2K^b/OVA_257–264 complex (MBL Co., Ltd., Nagoya, Japan). For intracellular cytokine staining, spleen T cells were incubated at a density of 2 × 10⁶ cells per mL in complete RPMI 1640 containing 10 μg/mL OVA_257–264 or OVA_323–339 for 24 h, and added with Golgi Stop (BD Biosciences) during the last 4–6 h. The cells were then harvested and stained by anti-CD4-APC and anti-IFN-γ-PE or anti-CD8-FITC and anti-IFN-γ-PE (BD Biosciences). Fluorescence profiles were acquired on a FACScan flow cytometer (Becton Dickinson) and analyzed using CellQuest software.

At the time of study enrollment, the patient characteristics were assessed; additionally, peripheral venous blood samples were taken and available for all study participants. All patients were enrolled in a stable condition free of any signs of congestion of acute cardiac ischemia. In patients presenting with ischemic HFrEF, the definition of heart failure was made at least 6 weeks after the acute ischemic event to overcome selection bias based on postinfarction myocardial stunning as recommended by the European Society of Cardiology [12 (link), 13 (link)]. Routine laboratory parameters were analyzed and processed according to the local standards of the Department of Laboratory Medicine of the Medical University of Vienna. In addition, cells from fresh EDTA blood samples were stained with APC-Cy7-conjugated Anti-CD4 (BD Biosciences, San Jose, CA, USA) and FITC-conjugated Anti-CD8. Regulatory T cells were identified via their intracellular forkhead-box protein P3 (Fox-P3) and CD25 expression using PE-conjugated Anti-Fox-P3 (BioLegend, San Diego, CA, USA) as well as APC-conjugated Anti-CD25 (BioLegend, San Diego, CA, USA) in a second FACS panel. Stained cells were analyzed using a BD FACS Canto II Flow Cytometer System and FACSDiva software.

The frequencies of Th17 cells and Th1 cells were analyzed as previously described [7 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from B-ALL patients and healthy donors were stimulated with 40 ng/ml phorbol 12-myristate13-acetate (PMA) and 1 μg/ml ionomycin for 5 h in the presence of 1 μg/ml brefeldin A (BFA; all from Sigma-Aldrich, St.louis, MO, USA). The cells were subsequently surface stained with a combination of PerCP-conjugated anti-CD3 and FITC-conjugated anti-CD8, fixed and permeabilized and intracellularly stained with PE-conjugated anti-IL-17A and APC-conjugated anti-IFN-γ (all from BD Biosciences, San Jose, CA, USA). Stained cells were acquired and analyzed using CellQuest software on a FACSCalibur instrument (BD Biosciences).