For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend. For in-vivo and ex-vivo studies, the following flow cytometry panel was used: anti-CD3 PE-Cy7, CD4 Pacific Blue, CD8 APC, CD44 PE, and anti-CD25 Alexa 700 all at a 1:200 dilution (except anti-CD25 at a 1:50 dilution) and were purchased from Biolegend. Endogenous Foxp3 expression was detected via eYFP fluorescence (or re-stained with anti-Foxp3 Efluor-450 clone FJK-16 s for any overnight staining (Fisher)), all acquisition was performed using a BD Fortessa flow cytometer. For surface FA transporter measurement, anti-CD36-APC (Biolegend), SLC27A1 (sigma), and SLC27A4 was purchased from Abcam (Cambridge, UK), followed by secondary goat anti-rabbit IgG Alexa-647 (Jackson Immunoresearch, West Grove, PA). Proliferation was analyzed using both FACS DIVA software as well as FlowJo software to determine expansion indexes.
Cd4 pacific blue
Manufactured by BioLegend
Sourced in United Kingdom
CD4-Pacific Blue is a fluorescent-labeled monoclonal antibody used for the detection and quantification of CD4-positive cells in flow cytometry analysis. It binds specifically to the CD4 surface marker expressed on T helper cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 apc, by BioLegend (4 mentions)
Facscanto 2, by BD (4 mentions)
Cd44 pe, by BioLegend (3 mentions)
Cd25 apc, by BioLegend (3 mentions)
Foxp3 af647, by BioLegend (3 mentions)
Tnf α apc, by BioLegend (3 mentions)
Hla dr percp cy5, by BD (3 mentions)
Cd8 apc cy7, by BioLegend (3 mentions)
Cd45ra apc, by BioLegend (3 mentions)
Cd4 pe cy7, by BioLegend (2 mentions)
Anti foxp3 efluor 450 clone fjk 16 s, by Thermo Fisher Scientific (2 mentions)
Anti cd36 apc, by BioLegend (2 mentions)
Slc27a1, by Merck Group (2 mentions)
Slc27a4, by Abcam (2 mentions)
Goat anti rabbit igg alexa 647, by Jackson ImmunoResearch (2 mentions)
Anti cd3 pe cy7, by BioLegend (2 mentions)
Anti cd25, by BioLegend (2 mentions)
Anti cd3 alexa700, by BioLegend (2 mentions)
Anti cd25 alexa 700, by BioLegend (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
24 protocols using cd4 pacific blue
1
Flow Cytometry Immunophenotyping Protocol
2
Flow Cytometry Immunophenotyping Protocol
3
SARS-CoV-2-Specific T-Cell Analysis
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Activation-induced marker (AIM) assays were performed as described by Reiss et al. [14 (link)]. The PBMC used in ELISpot assays were plated in round-bottom 96-well plates at a density of 1 × 106 cells/well in AIM-V medium, and incubated with medium alone as an unstimulated control, or with the AID SARS-CoV-2 peptide library—containing peptides from the S, N, M, and O proteins—for 18 h. Cells were blocked with TruStain (BioLegend, Amsterdam, The Netherlands) for 30 min; stained with CD4-Pacific Blue, CD134-FITC, CD25-PE-Dazzle, CCR7-PerCP-Cy5.5, and CD45RO-APC-Cy7 (BioLegend, Amsterdam, The Netherlands) for 60 min at 4 °C; and washed and analyzed on a Cytoflex S cytometer using CytExpert software. Cells were gated on the forward scatter/side scatter live cell gate, and then on the CD3+CD4+ gate for quantification of CD25hiCD134hi SARS-CoV-2-specific T-cells and memory T-cell subsets based on expression of CCR7 and CD45RO. All measurements are shown as background subtracted values (CD25hiCD134hi stimulated cells minus CD25hiCD134hi unstimulated cells as % total CD4+ T-cells). AIM assays were performed using the same flow cytometer with the same settings and CD25+CD134+ gate. Batches of antibodies were the same for all cohorts, with the exception of anti-CCR7, for which reason the CCR7 gate was adjusted relative to the CCR7+ naïve T-cell population.
4
PBMC Isolation and Activation Assay
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PBMCs from healthy donors were isolated by Ficoll gradient centrifugation from whole blood purchased from the New York Blood Center. Isolated cells were cultured in complete RPMI media, consisting of RPMI-1640 supplemented with 5 mM HEPES, 2 mM Glutamine, 50 μg/mL penicillin/streptomycin, 5 mM nonessential amino acids, 5 mM sodium pyruvate, and 10% fetal bovine serum (Thermo Fisher). PBMCs were incubated for 24 h in 6-well plates with IU1 (200 μM), b-AP15 (1 μM), or Romidepsin (0.1 μM). After washing with FACS buffer (PBS, 2% FBS), PBMCs were stained with the following cocktail of anti-human antibodies: CD3-FITC (BioLegend, San Diego, CA), CD4-Pacific Blue (BioLegend), CD25-PE-Cy7 (BioLegend), and CD69-Alexa Fluor 700 (BioLegend). In addition, LIVE/DEAD Yellow stain (Invitrogen, Thermo Fisher Scientific, Waltham, MA) was used to exclude dead cells. Staining was performed for 30 min on ice in FACS buffer. After washing, the samples were analyzed on a multi-laser flow cytometer LSR-II (BD Bioscience, San Jose, CA). Fluorescence compensation was performed with anti-mouse IgG (k) beads (BD Biosciences) stained with each antibody in a separate tube. The total content of PBMCs acquired by BD LSRII was analyzed with FlowJo (TreeStar, Cupertino, CA).
5
Flow Cytometric Analysis of Lymphocyte Subsets
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Cell suspensions from iliac lymph nodes, blood and tumors were first incubated with 1µg/106 cells of Fc block treatment (BD Pharmigen) and then stained for surface markers in FACS buffer (PBS + 1% FBS). For Treg staining on cells isolated from iliac lymph nodes: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD3 FITC, clone 145-2C11 (Biolegend); CD4 Pacific Blue, clone RM4-5 (Biolegend); Foxp3 APC, clone FJK-16S (eBioscience). For Treg staining on cells isolated from tumors: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD45 PerCP-Cyanine 5.5 (eBioscience); CD4 Pacific Blue, (Biolegend); CD8 Brilliant Violet 510, clone 53-6.7 (Biolegend); Foxp3 APC, (eBioscience). For effector memory staining on cells isolated from blood: Live/Dead (Fixable Near-IR); CD8 PerCP, (Biolegend); CD44 APC (Biolegend); CD62L FITC, clone MEL-14 (BD Pharmigen).
6
Comprehensive Treg Phenotyping by Flow Cytometry
7
Flow Cytometric Analysis of Regulatory T Cells
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The following anti-human antibodies were used: CD4-PE/Cy5, CD25-FITC, and Foxp3-Alexa647 from BioLegend (San Diego, CA, USA). Mouse cells were stained with fluorochrome-labeled mAbs to CD4-Pacific Blue, Brilliant Violet 605 and Alexa-700 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-APC (7D4), Ki-67-PE (Ki-67), and Thy1.1-PercP (Ox-7) from BioLegend. Foxp3-APC (FJK-16s), dead cell marker Viability Dye eFluor™ 780, and staining reagents from eBioscience (San Diego, CA, USA) were used according to the manufacturer’s instructions. To analyze expression of surface proteins, the cells were stained with the appropriate antibodies for 20 min at 4°C, washed once with FACS buffer (PBS, 0.1% BSA, and 0.02% NaN3), and fixed with 2% paraformaldehyde. For intracellular staining of Foxp3 and Ki-67, the cells were first surface stained, permeabilized with Fix/Perm (eBioscience), and stained with Foxp3 and Ki-67 antibodies diluted in Perm/Wash (eBioscience). To calculate absolute Treg numbers, unlabeled microbeads (BD Biosciences, Franklin Lakes, NJ, USA) were added to the stained cells and the following formula was used: absolute Treg numbers = (beads used × Treg events)/beads measured. Acquisition was performed on a BD™ LSR II or FACSCalibur, and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
8
Comprehensive Immune Profiling by Flow Cytometry
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CD4‐Pacific Blue (BioLegend, Cat.# 100531, clone: RM4‐5, 1:50), CD8a‐FITC (BioLegend, Cat.# 100706, clone: 53‐6.7, 1:50), CD45‐PerCP (BioLegend, Cat.# 103130, clone: 30‐F11, 1:80), CD279 (PD‐1)‐PE (BioLegend, Cat.# 135206, clone: 29F.1A12, 1:20), CD3‐APC/Cyanine7 (BioLegend, Cat.# 100222, clone: 17A2, 1:80), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).
9
Comprehensive Immune Cell Phenotyping
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For ex vivo analysis, 1-5×106 PBMC were cultured in 24-well plates for 3 hrs in the presence of PMA, ionomycin and GolgiStop (according to the manufacturer’s instructions; BD, Oxford, UK). Cells were stained for cell surface markers using CD3-PE-Cy7 (1:100) and CD14-APC-Cy7 (1:100) (BioLegend, Cambridge, UK), fixed in 2% paraformaldehyde, permeabilized with 0.5% Saponin and stained for CD4-PacificBlue (1:100) in combination with IL-17-PE (1:20), IFN-γ-PerCP-Cy5.5 (1:200), IL-10-AF488 (1:20), and TNF-α-APC (1:100) (all BioLegend). Co-cultures were re-stimulated at day three with PMA/ionomycin for 6 hrs, with GolgiStop present during the last 3 hrs. Cells were stained for cell surface markers using CD2-Pacific Blue (1:1,000) (BioLegend), CD14-APC-Cy7, fixed in 2% paraformaldehyde, and permeabilized with 0.5% Saponin and stained for IL-17-PE, IFN-γ-PerCP-Cy5.5, IL-10-AF488, and TNF-α-APC. Foxp3 was measured using Foxp3-AF647 (1:20) (BioLegend) according to manufacturer’s instructions. Monocyte phenotype was determined by staining with antibodies to CD14-APC-Cy7 (BioLegend), HLA-DR-PerCP-Cy5.5 (1:50) (BD), CD40-PE (1:50) (AbD Serotec, Kidlington, UK), and CD163-FITC (1:50) (SantaCruz, Santa Cruz, CA, USA). Cells were acquired using a FACSCantoII (BD) and analyzed using FlowJo software (TreeStar, Inc).
10
Comprehensive Immunophenotyping Panel
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Anti‐human GM‐CSF‐PE, CD3‐APC, CD33‐PE (Miltenyi Biotec, Auburn, CA); CD45RA‐Pacific blue, CCR7‐APC/cy7, CD4‐Pacific blue, CD8‐APC/cy7, CD19‐APC‐Cy7, CD3‐FITC, CD11b‐APC, CD38‐BV421, CD33‐BV605, CD34‐APC, CD45RA‐BV786 and CD16‐Pacific Blue (Biolegend, San Diego, CA); and CD116‐PE (BD Biosciences, Franklin Lakes, NJ) antibodies were used for analysis. Flow cytometric data were acquired by BD FACSCanto™II, BD FACSCelesta™ or BD Accuri™ C6 Plus (BD Biosciences San Jose, CA) and analysed by FlowJo (TOMY Digital Biology, Tokyo, Japan).
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