We analyzed the cell composition of the T cells specifically activated by SARS-CoV-2 in the IFN-γ assay by subtracting the basal cytokine response from the background control. We stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antibodies titrated to their optimal concentrations: CD45RA FITC (BD Pharmingen), CD27 APC (BD Pharmingen), CD3 VioGreen (Miltenyi Biotec), CD4 PECy7 (BD Pharmingen), CD8 APC Cy7 (BD Pharmingen), and 7AAD (BD Horizon). For the Treg panel CD25 BV421 (BD Horizon) and CD127 PE-CF594 (BD Horizon), were used. For the activation panel, HLA-DR BV 421 (BD Pharmingen), CD69 BV421 (Biolegend), and CD25 BV421 (BD Horizon), were used. For the exhaustion panel PD1 AF700 (Biolegend) and NKG2A BV421 (Biolegend) were used. For the chemokine panel, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) were used. Cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring an average of 200,000 cells. The analysis was performed using FlowJo 10.7.1 (FlowJo LLC).
Cd69 bv421
Manufactured by BioLegend
Sourced in United States
CD69-BV421 is a fluorophore-conjugated antibody that binds to the CD69 cell surface marker. CD69 is a type II transmembrane glycoprotein that is rapidly upregulated on the surface of activated lymphocytes. The BV421 fluorescent dye is used to label this antibody.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe cy7, by BioLegend (4 mentions)
Cd3 af700, by BioLegend (4 mentions)
Cd3 viogreen, by Miltenyi Biotec (2 mentions)
Ccr10 apc, by R&D Systems (2 mentions)
Cd45ra pe, by BioLegend (2 mentions)
Hla dr apc, by BioLegend (2 mentions)
Cd25 apc, by BioLegend (2 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd69 apc, by BioLegend (2 mentions)
Cd99 fitc, by BioLegend (2 mentions)
Ccr10 apc, by BD (2 mentions)
Mouse fitc cd4 and efluor 605 cd44, by BD (2 mentions)
Brilliant violet 570 cd4, by BioLegend (2 mentions)
Alexa fluor 700 cd44, by BioLegend (2 mentions)
Fitc klotho, by Bioss Antibodies (2 mentions)
Pe cy7 cd99, by Bioss Antibodies (2 mentions)
Pe cy5 vdr, by Bioss Antibodies (2 mentions)
Pe cy7 cnr2, by Bioss Antibodies (2 mentions)
Apc itga3, by R&D Systems (2 mentions)
13 protocols using cd69 bv421
1
SARS-CoV-2-Specific T Cell Activation
2
Multiparametric Flow Cytometry Immunophenotyping
3
Dissecting CD4+ T Cell Responses
4
Immunophenotyping of NK Cell Receptors
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We investigated the immunophenotyping of functionally relevant receptors within NK subpopulations by specific staining and posterior flow cytometry. We incubated purified NK cells (2 × 106 cells/mL) overnight with 100 ng/mL LPS and 10 µg/mL aCpG in the presence or absence of increasing concentrations of ruxolitinib (0.1 µM, 1 µM, 10 µM) at 37°C in complete media. After incubation, we performed immunophenotyping of functionally relevant receptors within the NK subpopulations with TIGIT PE-Vio770, CD69 BV421, TACTILE BV421 (Biolegend), CD56 AlexaFluor770, CD16 APC-Cy7, CD25 FITC, DNAM-1 PE, CXCR4 APC, NKG2D BV421, KIR3DL1 PE-Vio770, KIR2DL2/L3/S2 FITC, KIR3DL2 PE (BD Biosciences), NKp46 FITC, NKp44 PE, NKp30 APC, CD57 FITC, KIR2DS4 PE, NKG2A PE-Vio615, PD-1 FITC, LAG-3 PE, TIM-3 PE-Vio770, KIR2DL4 APC, KIR2DL1 BV421, CD3 Viogreen (Miltenyi Biotec), and NKG2C APC (R&D Systems). We acquired samples in a Navios flow cytometer (Beckman Coulter) and employed FlowJo v10.0.7 software (BD Biosciences) for the data analysis.
5
Granulocyte Profiling in Inflammatory Bowel Disease
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Blood granulocytes and single-cell suspensions obtained from digested biopsies of heathy donors and IBD patients were stained with the following antibodies CD66b PE-Cy7 (#305116 clone G10F5, 1/20 dilution, BioLegend), CD16 PerCP (#302028 clone 3G8, 1/20 dilution, BioLegend), CD62L APC-Cy7 (#304814 clone DREG-56, 1/20 dilution, BioLegend), CD69 BV421 (#310930 clone FN50, 1/20 dilution, BioLegend), CD193 FITC (#310720 clone 5E8, 1/20 dilution, BioLegend), CD63 FITC (#353005 clone H5C6, 1/20 dilution, BioLegend) and CXCR4 PE (# FAB170P clone 12G5, 1/10 dilution, R&D systems) and Zombie Aqua Fixable Viability Kit 1/1000 (#423101, BioLegend) for death cells. Cells were fixed using BD Stabilizing Fixative [BD], acquired using a BD FACSCanto II flow cytometer (BD) and analyzed with FlowJO software (version 10.6.1, BD).
6
Multiparametric T cell profiling
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The PBMC samples were recovered and stained against the following antibodies and their respective isotype controls: Anti-CD3-PerCP, Anti-CD4-BV510, Anti-CD8-APCCy7, CD69-BV421, PD-1-Alexa700, CLA-PE and CD103-APC (all from Biolegend, USA, see also the key resources table ). The cells were fixed by 1% paraformaldehyde (Sigma-Aldrich, USA) and analysed using CytoFlex (Beckman Coulter, USA). The data were exported and analysed by Flowjo v10.7.1 (TreeStar Inc, USA). Percentages of “cell positive” were normalized by the isotype controls of the same donors and time points.
7
Dissecting CD4+ T Cell Responses
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From the CCR10/CD99 co-stained sample, all four populations were isolated (CD99hiCCR10+, CD99loCCR10+, CD99loCCR10−, CD99hiCCR10−), although for some donors the CCR10+CD99lo population was too limited for subsequent stimulation assays. Sorted cells were collected in RPMI containing 20% FBS, washed once with PBS, counted and then resuspended at 10e6 cells/ml. About 400–500,000 sorted CD4 T cells were plated at a 1:2 ratio with CD4-depleted autologous PBMCs, such that the CD4 T cells represented in the well solely derived from the sorted fraction. This combined culture was subject to stimulation with: media only, 1 ul of 2017–2018 strain of flu vaccine (BEI resources), or 10 ug/ml HIV lysate (PepMix, JPT). After 17–19 hrs, cells were stained with CD69-BV421 (Biolegend), CD3-AF700 (Biolegend), CD4-PeCy7 (Biolegend), and eBio780 Fixable Viability Dye (eBio), fixed with 4% formaldehyde, and run on an LSRII.
8
Multiparametric Flow Cytometry Immunophenotyping
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Mouse FITC-CD4 and eFluor 605-CD44 [BD-Pharmingen]; KJ1.26 anti-TCR (Caltag Laboratories), Brilliant Violet 570-CD4 and Alexa Fluor 700-CD44 were from [Biolegend], FITC-Klotho,PE-Cy7-CD99, PE-Cy5-VDR, and PE-Cy7-CNR2 were from [Bioss]; APC-CCR10 and APC-ITGA3 were from [R&D Systems]; Fixable viability dye eFluor 780 was from [eBioscience]. Antibodies for human PBMC staining were as follows: from Biolegend, CD99-FITC,CD69-BV421 and CD69-APC , CD45R0-APC and CD45R0-BV421, CD3-AF700, CD4-Pe/Cy7, CD45RA-PE, Itga3 (CD49c)-PE, IL-7R-BV510, CD25-APC, HLA-DR-APC. From BD Biosciences, CCR10-APC and CCR10-PerCP Cy5.5. and viability dye eFluor 780 as above. Relevant mouse anti human isotype antibodies to IgG1k or IgG2ak were utilized for the following fluorophores: BV421, BV510, FITC, PE, and APC for both isotypes (Biolegend).
9
Flow Cytometric Analysis of Thymocytes
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Thymuses from WT or Yap-cKO mice (8–10 weeks old) were mechanically disrupted by being pushed through a 70 μm mesh (Falcon) with an insulin syringe plunger and washed with PBS. Cells were treated with ACK red blood cell lysis buffer, and thymocyte single-cell suspensions were stained for analysis by flow cytometry as described earlier. Cells were stained with dead cell dye and antibodies for the following surface markers: CD3 BUV737 (BD), CD4 BUV395 (BD), CD8 PerCP-Cy5.5 (Biolegend), TCRβ BV510 (Biolegend), CCR7 PECy7 (Biolegend), H-2Kb PE (Biolegend), CD69 BV421 (Biolegend), CD45R/B220 APCFire750 (Biolegend), CD25 APCFire750 (Biolegend), GL3 APCFire750 (Biolegend), and NK1.1 APCFire750 (Biolegend). Thymocytes were also stained intracellularly with anti-Nur77 APC (BD) using the eBioscience Foxp3/Transcription factor staining buffer set, as described earlier.
10
CD4+CTL Activation Assay in PBMCs
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