Pe conjugated anti foxp3

Cisplatin (purity 99%) and oxaliplatin (purity 99%) were purchased from Boyuan Chemical Company (Nantong, China) and dissolved in sterile water. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay kit and crystal violet were purchased from Sangon Biotech (Shanghai, China). ATP determination kit was purchased from Thermo Fisher (MA, United States). A cell apoptosis kit (PI-annexin V) was purchased from BD (NJ, United States). APC-conjugated calreticulin (CALR) antibody was purchased from Biorbyt (MO, United States). HMGB-1 antibody was purchased from Abcam (MA, United States). FITC-conjugated anti-CD83 (Clone: Michel-19), BV510 conjugated anti-CD86 (Clone: IT2.2), PerCP-Cy5.5 conjugated anti-CD8 (Clone: RPA-T8), BV421 conjugated anti-CD4 (Clone: OKT4), AF700 conjugated anti-IFN-γ (Clone: 4S.B3), and PE-conjugated anti-FoxP3 (Clone: 206D) were purchased from Biolegend (CA, United States). Vybrant™ DiD Cell-Labeling Solution was purchased from Invitrogen (CA, United States).

To determine the phenotypes of Th cells, 100 μL aliquots of blood were incubated with a Pacific blue-conjugated anti-CD4 (BD Biosciences) Ab, fixed, and then permeated for intracellular cytokine staining. The following Abs were used for intracellular cytokine staining: Alexa Fluor 488-conjugated anti-interleukin- (IL-) 4 (Biolegend), APC-conjugated anti-interferon- (IFN-) γ (BD Biosciences), and PE-conjugated anti-IL-17A (Biolegend) Abs. To analyze Treg, 100 μL aliquots of blood were incubated with Pacific blue-conjugated anti-CD4 and APC-conjugated anti-CD25 (eBioscience) Abs. After incubation for 30 min, leukocytes were fixed and permeated with Foxp3 staining buffer (eBioscience). PE-conjugated anti-Foxp3 (Biolegend) Abs were then added for the intracellular staining of Foxp3.
After staining, red blood cells were lysed, and leukocytes were analyzed by flow cytometry. CD4-positive lymphocytes were gated on the basis of low forward and side scatter. Phenotypes of Th cells are presented as percentages of Th-associated cytokine-expressing cells in CD4-positive lymphocytes. Treg populations are presented as a percentage of CD25/Foxp3 double-positive cells in gated CD4-expressing lymphocytes.

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.

To determine the phenotypes of Th cells, 100 μL aliquots of blood were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences), fixed and then permeated for intracellular cytokine staining. The following Abs were used for intracellular cytokine staining: Alexa Fluor 488-conjugated anti-IL-4 (Biolegend), APC-conjugated anti-interferon (IFN)-γ (BD Biosciences) and PE-conjugated anti-IL-17A (Biolegend) Abs. For analyzing the Treg subset, leukocytes were incubated with Pacific blue-conjugated anti-CD4 and APC-conjugated anti-CD25 (eBioscience) Abs. After incubation for 30 min, leukocytes were fixed and permeated with Foxp3 staining buffer (eBioscience). PE-conjugated anti-Foxp3 (Biolegend) Abs were then added to stain for intracellular Foxp3. After RBCs were lysed, the leukocytes were stained and analyzed by flow cytometry. CD4-positive lymphocytes were gated on the basis of low forward and side scatter. Phenotypes of Th cells are presented as percentages of Th-associated cytokine-expressing cells in CD4-positive lymphocytes. The Treg population is presented as a percentage of CD25/Foxp3 double-positive cells in gated CD4-expressing lymphocytes.