Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque™ PLUS (GE Healthcare, Sweden). Isolated PBMCs were resuspended in phosphate buffered saline (PBS) at a concentration of 1 × 106/ml and were stained with anti-human CD3-PEcy7 (eBiosciences, UK; clone: UCHT1), anti-human CD4 Alexa fluor 450 (eBiosciences, UK; clone: RPA-T4) and anti-human CD25 APC (Biolegend, UK; clone: BC96) antibodies for 20 min in the dark at 4 °C. Post incubation, cells were washed with PBS and re-suspended in Foxp3 Fix Perm Working solution (eBiosciences, UK) and incubated for 30 min in the dark at room temperature. Post incubation, cells were washed and resuspended in Foxp3 Permeabilization buffer (eBiosciences, UK) and stained with anti-human Foxp3 PE antibody (eBiosciences, UK) for 30 min in the dark at room temperature. Finally, the cells were washed and resuspended in PBS for flow cytometric analysis using a Cyan ™ ADP (Dako Ltd, UK). The percentage of CD3+ CD4+ CD25+ Foxp3+ T cells were recorded.
Apc anti human cd25
Manufactured by BioLegend
Sourced in United Kingdom
APC anti-human CD25 is a fluorochrome-conjugated antibody that binds to the CD25 antigen, also known as the alpha chain of the interleukin-2 (IL-2) receptor. CD25 is expressed on activated T cells, B cells, and natural killer cells. This antibody is commonly used in flow cytometry applications to identify and quantify CD25-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd3 pe cy7, by BioLegend (2 mentions)
Foxp3 permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Anti human cd3 pecy7, by Thermo Fisher Scientific (1 mentions)
Anti human cd4 alexa fluor 450, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Foxp3 fix perm solution, by Thermo Fisher Scientific (1 mentions)
Cyan adp flow cytometer, by Agilent Technologies (1 mentions)
Pe mouse igg1 κ isotype control, by BioLegend (1 mentions)
Apc mouse igg1 κ isotype control, by BioLegend (1 mentions)
Fluorescein isothiocyanate fitc anti human cd45ra, by BioLegend (1 mentions)
Apc anti human cd69, by BioLegend (1 mentions)
Apc anti human hla dr, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Brilliant violet 421 anti human cd4, by BioLegend (1 mentions)
Human foxp3 buffer set kit, by BD (1 mentions)
Percp cy5.5 anti human cd3, by BioLegend (1 mentions)
Pe anti human foxp3, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cell cycle staining kit, by Beyotime (1 mentions)
7 protocols using apc anti human cd25
1
Isolating and Analyzing Regulatory T Cells
2
Regulatory T Cell Phenotyping by Flow Cytometry
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PBMCs (1 × 106 cells/ml) resuspended in 50 µl of PBS were stained with anti-human CD3 PE cy7, anti-human CD4 Violet and anti-human CD25 APC (clone: PC61; Biolegend, UK) for 30 min at 4 °C. Post incubation, the cells were washed in PBS twice and fixed with Foxp3 Fix Perm solution (Thermo Fischer) for 30 min at room temperature, followed by a wash and staining with anti-human Foxp3 PE (clone: PCH101; Thermo Fischer) in diluted permeabilization buffer (Thermo Fischer) for 30 min at 4 °C. Post incubation, the cells were washed and analysed using a Cyan ™ ADP flow cytometer (Dako). Regulatory T cells were defined as CD3+ CD4+ CD25+ Foxp3+ cells [ gating strategy Supplementary Fig. 1 ] [32 (link)].
3
Activation Markers on Memory CD4+ T Cells
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The activation markers expression on resting memory CD4+ T cells were detected after IP-10 or CD3/CD28 stimulated and measured using the following fluorophore-conjugated antibodies: phycoerythrin (PE)-Cy7 anti-human CD3, allophycocyanin (APC)-Cy7 anti-human CD4, peridinin-chlorophyll-protein anti-human CD197/CCR7, fluorescein isothiocyanate (FITC) anti-human CD45RA, APC anti-human CD69, APC anti-human CD25, APC anti-human HLA-DR, APC mouse IgG1 κ isotype control, and PE mouse IgG1 κ isotype control (all from Biolegend). Samples were analyzed using an LSR II flow cytometer (BD Biosciences), which was adjusted with 10-peak color rainbow beads (Spherotech, Lake Forest, IL, USA). Gates were defined using appropriate isotype controls. Expression levels in each sample were analyzed with FlowJo v10 software (Ashland, OR, USA).
4
Isolation and Identification of Treg Cells
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Foxp3 is a specific marker of Treg. PBMC were separated from collected blood samples and Treg were detected by the expression of CD25 and Foxp3. We used the following fluorochrome-conjugated antibodies to stain the cell surface antigens: PerCp/Cy5.5 anti-human CD3, Brilliant Violet 421 anti-human CD4, APC anti-human CD25 (BioLegend, San Diego, CA, USA), and Ghost Rad780 Viability dye (Tonbo Bioscience, San Diego, CA, USA). We permeabilized cells using a Human Foxp3 buffer set kit (BD Bioscience, San Diego, CA USA), and stained Foxp3 using PE anti-human Foxp3 (BioLegend, San Diego, CA, USA). FCM was performed using BD FACS CantoII(Ver1.1, Diva6.1, BD Bioscience, San Diego, CA USA) and BD FACSDiva9, FlowJo (BD Bioscience, San Diego, CA USA) was used for data analysis. The gating strategy for the isolation of Treg involved the following steps: (1) isolation of live cells, (2) gating to isolate CD3- and CD4-positive cells, and (3) isolation of CD25- and Foxp3-positive cells (Supplementary Fig. 1 ).
5
Cell Cycle and Phenotypic Analysis of NSCLC and PBMCs
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Single-cell suspensions of NSCLC cells or PBMCs were stained with the cell cycle staining kit (Beyotime), FITC anti-human CD4, APC anti-human CD25, or PE anti-human Foxp3 antibodies (Biolegend). The stained cells were washed and incubated with 1 ml intracellular fixation buffer at room temperature for 60 min. Permeabilization buffer (2 ml) were then added into each tube, and the cells were centrifuged at 1,500 rpm for 7 min. The supernatants were removed, and single-cell suspensions were analyzed by flow cytometry (Beckman, China).
6
Chromatin Immunoprecipitation and T Cell Activation
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For the IP of P-TEFb, we used the following antibodies: anti-CDK9 (Abcam ab6544) or anti-CYCLIN T1 antibodies (Abcam; ab176702). For ChIP-qPCR for the detection of histone marks activation markers, we used anti-H3K27Ac (ab4729) and anti-H3K4me3 (ab8580). For detecting the different states of the phosphorylation of RNAPII CTD, we used the phosphorylated serine 2 antibody (Ser2P; ab238146) and phosphorylated serine 5 (Ser5P; ab5131). To monitor T cell activation following stimulation, the following antibodies were used: APC anti human CD25 (Biolegend; 302609); Pacific Blue anti human CD69 (Biolegend; 310919).
7
Activation of Human CD8+ T Cells
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Human peripheral blood CD8+ T cells (Stem Cell Technologies, 200–0164) were cultured in ImmunoCult XF T Cell Expansion media (Stem Cell Technologies, 10981) at 37 °C with 5% CO2. Antibodies were immobilized on Dynabeads M-280 Tosylactivated (Invitrogen, 14203) as described previously (Desai et al., 2022). Briefly, a total of 10 μg of antibody consisting of 2 μg of anti-human CD3 antibody (Bio X Cell, BE0001–2) and 8 μg of experimental antibody in 1 mL of 1X PBS was incubated at room temperature for 2 days with rotation. The beads were subsequently blocked with 10 mM glycine buffer for 1 h with rotation, followed by washing twice with PBSB. Next, the CD8+ T cells were plated at a density of 50,000 cells/well in a 96-well plate to a volume of 150 μL. Then, 50 μL of bead solution was added at a ratio of 4:1 or 8:1 of conjugated beads:T cells. After incubating at 37 °C with 5% CO2 for 96 h, the cells were stained with BV510 anti-human CD8 (BioLegend, 344732) and APC anti-human CD25 (BioLegend, 302610) antibodies at 1:200 dilution each on ice for 5 min. Cells were then centrifuged at 300 xg for 4 min, washed with 1X PBS, and analyzed via flow cytometry.
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