Abs against following antigens: CD3-FITC, CD3-APC, CD25-FITC, CD4-PE, CD4-PECy7, CD8-PE, B220-PE, NK-1.1-PE, CD69-APC, FOXP3-PE, IFN-γ-APC, Isotype-matched control Ab, Annexin V-FITC, 7AAD, Purified anti-CD25 mAb (PC61) were purchased from BioLegend (San Diego, CA). The murine recombinant cytokines (IL-2, IL-12) were from Peprotech (Rocky Hill, NJ).
Cd4 pe cy7
Manufactured by BioLegend
Sourced in United States, United Kingdom
CD4-PE-Cy7 is a fluorescence-conjugated antibody used for flow cytometry analysis. It binds to the CD4 antigen expressed on the surface of T helper cells. The PE-Cy7 fluorescent dye is used for signal detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BioLegend (10 mentions)
Cd8 apc, by BioLegend (10 mentions)
Cd19 apc, by BioLegend (8 mentions)
Cd3 apc, by BioLegend (7 mentions)
Cd19 apc cy7, by BioLegend (7 mentions)
Cd8 pe, by BioLegend (6 mentions)
Cd44 pe, by BioLegend (6 mentions)
Foxp3 pe, by BioLegend (6 mentions)
Cd45ro apc, by BioLegend (5 mentions)
Cd44 fitc, by BioLegend (5 mentions)
Cd45 fitc, by BioLegend (5 mentions)
Cd3 percp, by BioLegend (4 mentions)
Cd45ra pe, by BioLegend (4 mentions)
Cd99 fitc, by BioLegend (4 mentions)
Ccr10 apc, by BD (4 mentions)
Cd69 bv421, by BioLegend (4 mentions)
Cd3 af700, by BioLegend (4 mentions)
Cd8b precp cy5, by BioLegend (4 mentions)
Cd45 buv395, by BD (4 mentions)
Cd8 percp cy5, by BioLegend (4 mentions)
97 protocols using cd4 pe cy7
1
Multicolor Flow Cytometry Panel
2
Pleural Fluid Chemotaxis Assay
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Whole blood was lysed with RBC lysing solution (BioLegend). Cells were washed and the cell count was performed by MACSQuant cytometer. Cells were resuspended in RPMI 1640 with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Biowhittaker, Verviers, Belgium) at 2 × 106 cells/ml. In 24 plate-wells, 500 μl of RPMI 1640 with 10% fetal calf serum or 0.22 μm filtered medium RPMI supplemented with 10% of pleural fluid from LAC was added to the wells. 3 × 105 cells were added in 3 μm pore-size filters (Millipore Corporation, Billerica, MA) and incubated for 4 h at 37ΊC. After culture, healthy donor cells that migrated towards the medium or pleural fluids were collected from the wells, washed with PBS and stained with anti-CD3-PECy5, CD4-PECy7 (BioLegend) and CD20-FITC (Immunotools) monoclonal antibodies for 20 min. Cells were washed and resuspended in 300 μl of PBS to be acquired by flow cytometry. To analyze the migration fold of cells towards the pleural fluids, we calculated the relative cell migration that is the ratio between the absolute numbers of CD4+ T, CD8+ T cells and CD20+ B cells that migrate to the medium supplemented with pleural fluid and the absolute cell numbers of each respective subset that migrate to the medium.
3
Isolation and Analysis of Immune Cells from Lung and Spleen
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Lung tissue was chopped and digested using collagenase D (Roche Diagnosis) in Dulbecco’s modified Eagle’s medium (Life Technologies, Carlsbad, CA) for 30 min at 37 °C with agitation. Next, the chopped lung or spleen tissue was passed through a 40-μm cell strainer to obtain single-cell suspensions before RBC lysis. Cells were incubated with LIVE/DEAD Aqua (Thermo Fisher Scientific). Cells were stained with the following monoclonal antibodies: anti-mouse CD45-PerCP-Cy5.5 (BioLegend, San Diego, CA, USA), CD4-PE-Cy7 (BioLegend), CD25-PE (eBioscience), and Foxp3-APC (eBioscience, Waltham, MA, USA). Flow cytometry analysis was performed using a BD FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA), and the results were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
4
Flow Cytometry Assay for CD4+ and CD8+ T Cell Responses
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CD4+ and CD8+ T cell responses were measured from blood and tissues by flow cytometric ICS, as previously described.54 (link) Briefly, 1 × 106 mononuclear cells were incubated with Gag or Vif open-reading frame pools and the co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by addition of Brefeldin A (Sigma-Aldrich) for an additional 8 h. Co-stimulation without antigen served as a background control, while incubation with Staphylococcal Enterotoxin B (Toxin Technology) served as the positive control. The cells were then labeled with CD4 PE-Cy7 (Biolegend) and CD8 PerCP-Cy5.5 (BD Biosciences) and fixed with 2% paraformaldehyde. After permeabilization, the cells were stained with CD3 Pacific Blue, IFN-γ APC, TNF-α FITC (BD Biosciences, all), and CD69 PE-Texas Red (Beckman Coulter). The cells were fixed and flow cytometric analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star, Ashland, OR). In some cases, cells were CD25-depleted prior to setting up the ICS experiment to remove T regulatory cells (Miltenyi Biotec).
5
Multicolor Leukocyte Immunophenotyping in Mice
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For leukocyte immunostaining, mouse blood, spleen and colon samples were obtained to prepare single cell suspensions. The fixable viability dye eFluor® 780 (eBioscience, Thermo Fisher Scientific, MA) was used to label dead cells. To block nonspecific binding of Fc receptors, cells were pre-incubated with a purified anti-CD16/32 antibody for 10 min on ice prior to immunostaining. Then, the cells were stained with the appropriate combination of the following fluorochrome-conjugated mAbs against mouse CD11b (FITC), CD3ε (FITC), CD8α (PE), NK1.1 (PE), F4/80 (PE), Gr-1 (APC), CD19 (APC), CD4 (PE/CY7) or C5aR-1 (PE) (Biolegend, San Diego, CA) on ice for 30 min according to the manufacturer's instructions, in which the fluorochrome-conjugated isotype-specific IgGs were treated as controls to determine the quadrants and gates. All samples were assayed by a BD FACSCantoTM II Cell Analyzer (BD Biosciences, San Jose, CA), and the data were analyzed with FlowJo software (Tree Star, Ashland, OR).
6
Immunophenotyping of PBMC Populations
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PBMC were washed and maintained in MACS buffer (Miltenyi Biotech, Auburn, CA, USA) during the staining protocol for specific cell markers. Cells were stained first with viability marker according to the manufacturer’s instructions (Thermo Fisher Scientific LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Eugene, OR, USA) followed by incubation with an FcR block (BD Pharmingen) and, afterward, with specific Abs to the corresponding extracellular markers such as CD3-FITC and CD4-PE-Cy7 (both from BioLegend, San Diego, CA, USA), and CD25-allophycocyanin (eBioscience) were applied. To stain for intracellular markers, the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences) was used as described previously with anti-Foxp3-eFluor 450 Ab (Invitrogen) [17 (link)]. The appropriate isotype control Abs were applied to separate control tubes as described and the staining profile was used to establish the negative gate [17 (link)]. After a final wash, either 10,000 cells/tube or 30,000 cells/tube out of 1 × 106 cells/tube placed in MACS buffer were acquired in logarithmic scales and analyzed with the BD LSR Fortessa flow cytometer. FlowJo software was used for the live cell gating and their further analysis.
7
Flow Cytometry Immunophenotyping Protocol
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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.
8
CFSE-Labeled PBMC Proliferation Assay
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PBMCs were incubated with 1.25 μl/ml CFSE (Invitrogen) at 37°C. After 10 min, the cells were washed twice at 4°C with 5 ml Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum (FBS). Cells were then resuspended in 600 μl of RPMI/10% FBS and seeded into 96-well plates along with 5 μg/ml phytohemagglutinin (PHA), 10 μg/ml lectin from pokeweed mitogen (PWM), and the same volume of RPMI for 72 h. After staining with CD3-PerCP (clone: HIT3a, BioLegend), CD4-PE-Cy7 (clone: RPA-T4, BioLegend), CD8-PE (clone: RPA-T8, BioLegend), and CD19-APC (clone: HIB19, BioLegend) antibodies, cells were analyzed and examined by flow cytometry (8 (link)).
9
Isolation and Sorting of Resting CD4 T Cells
10
Flow Cytometry Immunophenotyping Protocol
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