Cell surface and intracellular staining were conducted as previously described (28 (link)). The experiments used the following reagents: CFDA-SE, PE-conjugated mouse anti-human IFN-γ (BD Pharmingen), APC-conjugated rat anti-human IL-2 (BD Pharmingen), PE-Cy™7-conjugated mouse anti-human TNF-α (BD Pharmingen), APC-conjugated rat anti-human IL-10 (BD Pharmingen), FITC-conjugated mouse anti-human CD4 (BD Pharmingen), Alexa Fluor® 647-conjugated mouse anti-human Foxp3 (BD Pharmingen), APC-conjugated mouse anti-human CD4 (BioLegend), APC-conjugated mouse anti-human CD8 (BioLegend), PE-conjugated mouse anti-human PD-1 (BD Pharmingen), FITC-conjugated Annexin V (BD Pharmingen), and an Annexin V PE Apoptosis Detection Kit I (BD Pharmingen) with the matched isotype controls. Fluorescence was evaluated via FACS analysis using a BD Accuri™ C6 system within one hour of cell staining. The data were analyzed using FlowJo 7.6 software (Tree Star).
Fitc conjugated mouse anti human cd4
Manufactured by BD
Sourced in United States
The FITC-conjugated mouse anti-human CD4 is a monoclonal antibody conjugated with fluorescein isothiocyanate (FITC). It is designed to detect and quantify the CD4 protein on human cells.
Automatically generated - may contain errors
Lab products found in correlation
Melatonin, by Merck Group (2 mentions)
Pe conjugated mouse anti human ifn γ, by BD (1 mentions)
Accuri c6 system, by BD (1 mentions)
Apc conjugated mouse anti human cd4, by BioLegend (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Fitc conjugated annexin 5, by BD (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Goldiplug, by BD (1 mentions)
Ionomycin, by BioLegend (1 mentions)
Human th17 magnetic bead panel, by Merck Group (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
4 protocols using fitc conjugated mouse anti human cd4
1
Cytokine and Apoptosis Analysis by Flow Cytometry
2
WT1-specific helper T cell induction
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The induction of WT1-specific helper T cells was also measured. Human PBMCs positive for HLA-DRB1*14:54/15:01, HLA-DQB1*05:03/06:03, and HLA-DPB1*04:01/04:02were incubated for 10 days with adegramotide (Bachem). On Day 10, 40 μg/mL adegramotide or 0.125% DMSO were added and incubated for 3.5 h. Following an overnight incubation with GoldiPlug (BD Biosciences), cells were stained for CD4 (FITC-conjugated mouse anti-human CD4; BD Biosciences), fixed using a Fixation/Permeabilization Kit (BD Biosciences), and stained with phycoerythrin-conjugated mouse anti-human interferon-gamma (IFN-γ) (BD Biosciences).
3
Melatonin Modulates Immune Cell Cytokines
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PBMCs were stimulated with phorbol myristate acetate (PMA), ionomycin and brefeldin A (423304, BioLegend) in the presence of 10-4 M melatonin (Millipore, Sigma) or vehicle (0.02% dimethyl sulfoxide). Six hours later, the activated cells were stained with FITC-conjugated mouse anti-human CD4 (550628, BD) at 4°C for 30 minutes. After surface staining, the cells were fixed and permeabilized for 1 hour at room temperature by BD Cytofix/Cytoperm buffer (BD, USA). Intracellular cytokine staining was performed with APC-conjugated mouse anti-human IL-10 (1:20, 501410, BioLegend), PE-conjugated mouse anti-human IL-17 (1:5, 560438, BD), PerCP-Cy5.5-conjugated rat anti-human IL-6 (1:20, 561118, BioLegend), PE-conjugated mouse anti-human TNFα (1:10, 559321, BD), APC-conjugated mouse anti-human IFNγ (1:10, 551385, BD), and PerCP-Cy5.5-conjugated mouse anti-human IL-9 (1:20, 507610, BioLegend). For cytokine measurement, the supernatant of unstimulated or melatonin-stimulated (48 hours) cell cultures was collected and assessed for nine cytokines (IFNγ, TNFα, IL-2, IL-4, IL-6, IL-9, IL-17A, IL-22, and IL-21) using Luminex technology (Human TH17 Magnetic Bead Panel, Merck, Germany).
4
Immunophenotyping of Peripheral Blood Cells
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Peripheral blood (2 ml) was taken before surgery, on day 0 (before initiation of first round of chemotherapy) and day 20 (before initiation of second round of chemotherapy). Blood samples were collected in sterile heparinized vials and analysed immediately. Aliquots of heparinized whole blood (100 µl) were incubated for 15 min at room temperature with antibodies (all BD Biosciences, San Jose, CA, USA): (i) FITC-conjugated mouse antihuman CD4, APC-conjugated mouse antihuman CD25 and PE-conjugated mouse antihuman FOXP3; (ii) FITCconjugated mouse antihuman CD8 and PE-conjugated mouse antihuman CD28; or (iii) FITC-conjugated mouse antihuman CD3 and PE-conjugated mouse antihuman CD16/CD56. Erythrocytes were lysed using lysing solution and washed twice with phosphate-buffered saline (pH 7.4), containing 2% fetal calf serum and 2% sodium azide. Intranuclear staining of FOXP3 was performed according to the manufacturer's instructions (BD Biosciences). Cells were analysed by FACSCalibur™ (BD Biosciences), and the resulting data were analysed using CellQuest™ software. A minimum of 5000 gated events/condition were analysed. CD4 + CD25 + FOXP3 + cells were quantified as a percentage of CD4 + cells, CD8 + CD28 -cells were quantified as a percentage of CD8 + cells, and NK (CD3 -CD16 + CD56 + ) cells were quantified as a percentage of lymphocytes.
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