Anti cd4 alexa fluor 700

Manufactured by BioLegend

Sourced in United States, United Kingdom

Anti-CD4-Alexa Fluor 700 is a fluorescently labeled antibody that binds to the CD4 antigen expressed on the surface of T helper cells. It is designed for use in flow cytometry applications to identify and quantify CD4-positive cell populations.

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Lab products found in correlation

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Close spelling variants of this product

Anti-CD4 (192 citations) Alexa Fluor 700 (29 citations) CD4-Alexa Fluor 700 (15 citations) CD4-Alexa 700 (4 citations)

9 protocols using anti cd4 alexa fluor 700

Multiparametric FACS Analysis of T Cells

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For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.

Wiese T., Dennstädt F., Hollmann C., Stonawski S., Wurst C., Fink J., Gorte E., Mandasari P., Domschke K., Hommers L., Vanhove B., Schumacher F., Kleuser B., Seibel J., Rohr J., Buttmann M., Menke A., Schneider-Schaulies J, & Beyersdorf N. (2021). Inhibition of acid sphingomyelinase increases regulatory T cells in humans. Brain Communications, 3(2), fcab020.

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Multicolor Flow Cytometry Immunophenotyping

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The following mouse flow cytometry conjugated antibodies were used at the concentration suggested on the manufacturer’s data sheet for surface or intracellular staining: Live/Dead Ghost UV450, anti-CD45 BUV805, anti-PD1 BV421, anti-CD8 BV570, anti-NKG2D FITC, anti-CD278 BV785, anti-IFNγ PerCP-Cy5.5, anti-PD-L1 PE-Dazzle 594, anti-NK1.1 Pe-CY5, anti-CD3 Pe-Fire700, anti-CD4 AlexaFluor700, anti-Ki-67 Pacific Blue, anti-FoxP3 Alexa532, anti-Granzyme B PE, anti-IL-17A APC all purchased from Biolegend or Thermofisher. For intracellular staining of FoxP3, Granzyme B, IL-17A, and Ki-67, the cells were fixed and permeabilized using cold 70% ethanol. Immuno-stained cell percentages were assessed by a Cytek Aurora flow cytometer and analyzed by Flow-Jo software.

Singh K.V., Malathum K, & Murray B.E. (2001). In Vitro Activities of a New Ketolide, ABT-773, against Multidrug-Resistant Gram-Positive Cocci. Antimicrobial Agents and Chemotherapy, 45(12), 3640-3643.

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Comprehensive Immune Profile of PBMCs

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After resting 2-3h in complete medium at 37°C, thawed PBMC were stained with the following mAbs: anti-CD4-Alexa Fluor 700 (BioLegend Inc., San Diego, CA, USA), anti-CD8-PE-Texas Red (Invitrogen), anti-CD45RA-Qdot655, anti-CCR7-BV421, anti-HLA-DR-V500 (BD Biosciences), and anti-PD-1-BV711 (BioLegend). Cells were subsequently fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), stained with anti-Ki67-PerCP eFluor710 (eBioscences, San Diego, CA) and anti-perforin-FITC (Diaclone, Besançon, France), and acquired with a LSR II Fortessa flow cytometer (BD Biosciences). Analysis was performed with FlowJo software (FlowJo LLC, Ashland, OR); after gating on total memory CD4⁺ and CD8⁺ lymphocytes, the percentage of IL-7R^pos and IL-7R^neg cells was calculated, as well as the percentage of IL-7R^pos and IL-7R^neg CD4⁺ T cells expressing Ki67, HLA-DR, PD-1 and perforin.

Mele F., Fornara C., Jarrossay D., Furione M., Arossa A., Spinillo A., Lanzavecchia A., Gerna G., Sallusto F, & Lilleri D. (2017). Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission. PLoS ONE, 12(11), e0187731.

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Comprehensive T Cell Phenotyping in AAV

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We obtained PBMCs from 10 patients with AAV with a high BVAS (BVAS > 15) and 10 patients with AAV with a low BVAS (BVAS < 5) and analyzed the surface expression of PD-1, Tim-3, and CD28 on T cells using flow cytometry. The following antibodies were used for staining: anti-CD3-V500 (BD Biosciences, Oxford, UK), anti-CD4-Alexa Fluor 700, anti-CD25-APC, anti-CD28-PE-Cy7, anti-CD279 (PD-1)-BV421, and anti-CD388 (Tim-3)-PE ( BioLegend, CA, USA). Lymphocytes were gated using forward and side scatter parameters, and samples were analyzed using FACSVerse (BD Biosciences, Oxford, UK) and associated software programs (FlowJo).

Pyo J.Y., Yoon T., Ahn S.S., Song J.J., Park Y.B, & Lee S.W. (2022). Soluble immune checkpoint molecules in patients with antineutrophil cytoplasmic antibody-associated vasculitis. Scientific Reports, 12, 21319.

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Flow Cytometry Analysis of Activated T-Cell Phenotypes

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For surface marker staining, cells were stained with antibodies for 15 minutes at room temperature. For ex vivo activation, cells were stimulated with a stimulation cocktail (51-2042E; BD Biosciences, Franklin Lakes, NJ) and simultaneously stained with fluorochrome-coupled anti-CD107a antibody. Dead cells were excluded by using a live/dead cell staining kit (ThermoFisher scientific, Waltham, MA). The following antibodies were used for flow cytometry analysis: anti–CD3-Brilliant violet (BV) 605 (clone 17A2; Biolegend, San Diego, CA), CD8-Alexa Fluor 700 (clone 53-6.7; Biolegend), anti-CD8-Pacific Blue (PB) (clone 53-6.7; Biolegend), anti–CD44-Phycoerythrin (PE)/Cy7 (clone 1M7; Biolegend), anti-CD62L PerCP/Cy5 (clone MEL-14; Biolegend), anti–CD4-Alexa Fluor 700 (clone GK1.5; Biolegend), anti–IFNγ- Allophycocyanin (APC) (clone XMG1.2; Biolegend), anti–CD107a–PE (clone 1D4B; BD Biosciences), anti–MHC-I–APC (clone 28-8-6; Biolegend), anti–MHC-I–APC (clone W6/32; Invitrogen, Carlsbad, CA), and anti–PD-L1–PerCP/Cy5.5 (clone 10F.9G2; Biolegend).

Wabitsch S., Tandon M., Ruf B., Zhang Q., McCallen J.D., McVey J.C., Ma C., Green B.L., Diggs L.P., Heinrich B, & Greten T.F. (2021). Anti–PD-1 in Combination With Trametinib Suppresses Tumor Growth and Improves Survival of Intrahepatic Cholangiocarcinoma in Mice. Cellular and Molecular Gastroenterology and Hepatology, 12(3), 1166-1178.

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Flow Cytometry Antibody Panel

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Antibodies used for flow cytometry were purchased from BioLegend (San Diego, CA) and include: anti‐CD4‐Alexa Fluor 700 (RPA‐T4), CD69‐APC/Cy7 (FN50), and CD25‐PE (M‐A251).

Holbrook A.K., Peterson H.D., Bianchi S.A., Macdonald B.W., Bredahl E.C., Belshan M, & Siedlik J.A. (2019). CD4+ T cell activation and associated susceptibility to HIV‐1 infection in vitro increased following acute resistance exercise in human subjects. Physiological Reports, 7(18), e14234.

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Multiparametric Immune Cell Profiling

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Cells were surface-stained with anti-CD3-FITC, anti-CD4-AlexaFluor700, anti-CD8-PE/Dazzle594, anti-CD127-PE-Cy7, anti-CCR6-BrilliantViolet421, anti-CCR4-PE, and anti-CXCR3-PerCP/Cy5.5 (Biolegend). Cells were fixed and permeabilized to stain for the intracellular transcription factor FoxP3 using anti-FoxP3-AlexaFluor647 according to manufacturer’s instructions (eBioscience). Cell fluorescence was measured using a LSR II Fortessa flow cytometer (BD Biosciences) and analysis was performed using FlowJo software (Tree Star).

Adegunsoye A., Hrusch C.L., Bonham C.A., Jaffery M.R., Blaine K.M., Sullivan M., Churpek M.M., Strek M.E., Noth I, & Sperling A.I. (2016). Skewed Lung CCR4 to CCR6 CD4+ T Cell Ratio in Idiopathic Pulmonary Fibrosis Is Associated with Pulmonary Function. Frontiers in Immunology, 7, 516.

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Comprehensive Immunophenotyping of Mouse Immune Cells

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The following antibodies were utilized for flow cytometric analysis of mouse splenocytes or bone marrow cells: PacBlue-anti-B220 (RA3-6B2); Alexa Fluor 700-anti-CD4 (RM4-5); PE-anti-PD-1 (29F.1A12); PerCP-Cy5.5-anti-CD69 (H1.2F3); APC-Cy7- anti-CD25 (PC61); Cy5-anti-CD86 (GL1); PeCy7-anti-CD95 (FAS, Jo2); PeCy7-anti-MHC-II (M5/114.15.2); APC-anti-CD24 (HSA) (M1/69); Biotin-anti-Ly5.1 (BP-1) (6C3); FITC-anti-CD23 (B3B4); PE-Cy5-streptavidin (SA) were from purchased from BioLegend, San Diego, CA. Biotin-anti-CXCR5 (2G8) from BD Pharmingen, San Diego, CA. FITC-peanutagglutinin (PNA) from Vector Labs, Burlingame, CA. PE-anti-IgM (eB121-15F9); APC anti-CD93 (AA4.1); FITC-anti-F4/80 (BM8) from eBiosciences, San Diego, CA. The following antibodies were utilized for phosphoflow analysis of purified mouse B cells: PE-anti-Btk (pY223)/ItK (pY180) (N35-86); Alexa Fluor 647-anti-ERK1/2 (pT202/pY204) (20A); Pacific Blue-anti-p38 MAPK (pT180/pY182); Alexa Fluor 488-anti-Syk (pY348) (I120-722). Stained cells were analyzed using the BD LSR II flow cytometer (BD Biosciences, Franklin lakes, NJ). Data were acquired using FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA).

Wong E.B., Soni C., Chan A.Y., Domeier P.P., S.h.w.e.t.a.n.k., Abraham T., Limaye N., Khan T.N., Elias M.J., Chodisetti S.B., Wakeland E.K, & Rahman Z.S. (2015). B cell-intrinsic CD84 and Ly108 maintain germinal center B cell tolerance. Journal of immunology (Baltimore, Md. : 1950), 194(9), 4130-4143.

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Flow Cytometry of Mouse Immune Cells

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The following antibodies were utilized for flow cytometric analysis of mouse splenocytes or bone marrow cells: PacBlue-anti-B220 (RA3-6B2); Alexa Fluor 700-anti-CD4 (RM4-5); PE-anti-PD-1 (29F.1A12); PerCP-Cy5.5-anti-CD69 (H1.2F3); APC-anti-TCR Vα2 (B20.1); APC-Cy7- anti-CD25 (PC61); Cy5-anti-CD86 (GL1); PeCy7-anti-CD95 (FAS, Jo2); PeCy7-anti-MHC-II (M5/114.15.2); APC-anti-CD24 (HSA) (M1/69); Biotin-anti-Ly5.1 (BP-1) (6C3); FITC-anti-CD23 (B3B4); PE-Cy5-streptavidin (SA) were from purchased from BioLegend, San Diego, CA. Biotin-anti-CXCR5 (2G8); FITC-anti-CD11c (HL3); FITC-anti-CD43 (S7) from BD Pharmingen, San Diego, CA. FITC-peanut-agglutinin (PNA) from Vector Labs, Burlingame, CA. PE-anti-IgM (eB121-15F9); APC-anti CD93 (AA4.1); FITC-anti-F4/80 (BM8) from eBiosciences, San Diego, CA. Stained cells were analyzed using the BD LSR II flow cytometer (BD Biosciences, Franklin lakes, NJ). Data were acquired using FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA). Dead cells were quantified by flow cytometry using 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO).

Soni C., Wong E.B., Domeier P.P., Khan T.N., Satoh T., Akira S, & Rahman Z.S. (2014). B cell-intrinsic TLR7 signaling is essential for the development of spontaneous germinal centers. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4400-4414.

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