Cells from animals were stained with fluorescently conjugated antibodies: CD69-Brilliant Violet 510 (clone FN50) or CD14-Brilliant Violet 510 (clone M5E2), CD3-Pacific Blue (clone Hit3a), CD8a-FITC (clone Hit8a), CD4-PE (clone RPA-T4), CD19-PE-Cy5 (clone SJ25C1), CD45-APC (clone 2D1), CD56-PE-Cy7 (clone MEM-188) (all from Biolegend) and Ghost Dye Red 780 (Tonbo Biosciences). All flow cytometry samples were run using a MACSQuant Analyzer 10 flow cytometer (Miltenyi) or Attune NxT. (Beckman Coulter). All data was analyzed using FlowJo v.10 (TreeStart, Inc) (Supplementary Fig. 10d ).
Cd4 pe clone rpa t4
Manufactured by BioLegend
CD4-PE (clone RPA-T4) is a fluorescently labeled antibody that binds to the CD4 surface protein. It is a tool used for the identification and analysis of CD4-positive cells in flow cytometry applications.
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Lab products found in correlation
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Cd69 brilliantviolet510, by BioLegend (1 mentions)
Cd14 brilliant violet 510, by BioLegend (1 mentions)
Cd45 apc clone 2d1, by BioLegend (1 mentions)
Attune nxt, by Beckman Coulter (1 mentions)
Cd8a fitc, by BioLegend (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Human fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
2 protocols using cd4 pe clone rpa t4
1
Multiparameter Flow Cytometry Immunophenotyping
2
Characterization of T Cell Subsets by Flow Cytometry
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To determine the expression of cell surface markers, cells were blocked with human Fc receptor blocking reagent (Miltenyi Biotec) and then stained with fluorochrome-conjugated antibodies diluted in flow cytometry staining buffer (PBS containing 2% FBS). To stain for intracellular transcriptional factors, Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) was used. The following fluorochrome-conjugated antibodies were applied: anti-CD3-eFlour450 (clone UCHT1, Thermo Fisher Scientific), CD4-PE (clone RPA-T4, Biolegend), CD4-FITC (clone RPA-T4, Thermo Fisher Scientific), T-bet-FITC (clone ebio4B10, Thermo Fisher Scientific), GATA-3-AF488 (clone TWAJ, Thermo Fisher Scientific), ROR-γT-PE (clone AFKJS-9, Thermo Fisher Scientific), Foxp3-FITC (clone 150D/E4, Thermo Fisher Scientific). For flow cytometry, we have adhered to the guidelines for the use of flow cytometry and cell sorting in immunological studies [71] . The gating strategy is shown in Supporting information Figure S8.
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