Pacific blue conjugated anti cd4

Manufactured by BD

Pacific blue-conjugated anti-CD4 is a fluorescently labeled antibody that binds to the CD4 receptor on the surface of T lymphocytes. It is designed for use in flow cytometry applications to identify and quantify CD4-positive cells in a sample.

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Lab products found in correlation

Foxp3 staining buffer, by Thermo Fisher Scientific (2 mentions) Apc conjugated anti cd25, by Thermo Fisher Scientific (2 mentions) Pe conjugated anti foxp3, by BioLegend (2 mentions) Pe conjugated anti il 17a, by BioLegend (2 mentions) Apc conjugated anti interferon ifn γ, by BD (2 mentions) Pacific blue conjugated anti cd4, by Thermo Fisher Scientific (2 mentions) Pe conjugated anti cd11b, by BD (1 mentions) Percp conjugated anti cd8, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Alexa700 conjugated anti cd3, by BD (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Apc h7 conjugated anti cd8, by BD (1 mentions) Facsdiva v8, by BD (1 mentions) Kaluza v1, by Beckman Coulter (1 mentions) Facsaria 3, by BD (1 mentions)

5 protocols using pacific blue conjugated anti cd4

Phenotypic Profiling of Pancreatic Lymph Nodes

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Pancreatic lymph nodes (pLN) and spleen were harvested from NOD8.3 mice and prepared as single-cell suspensions. Cell surface markers were assessed by staining the cells with pacific-blue conjugated anti-CD4 (558107), PerCP conjugated anti-CD8 (561092), to allophycocynin conjugated anti-CD44 (559250), fluorescein isothiocyanate conjugated anti-CD62 (561917), PECy7 conjugated anti-CD25 (552880), PECy7 conjugated CD19 (552854), APCCy7 conjugated anti-B220 (552094), PE conjugated anti-CD11b (553311), Pacific Orange-conjugated streptavidin against biotin-conjugated anti-CD11c (553800) (all antibodies 1:400; BD Bioscience). Intracellular staining with Foxp3 (560409; BD Biosciences) was performed according to the manufacturer's instructions. Flow cytometry was performed on FACSCanto II (BD Bioscience, San Jose, CA, USA) and data was analyzed with Flowjo analysis software (v10.0.8; BD Bioscience). Cells were gated on total lymphocytes and the plot was either gated on CD8⁺ and CD4⁺, CD19⁺ and B220⁺, or quadrant gates for CD11c and CD11b to determine CD11c⁺CD11b⁺ and CD11c⁺CD11b⁻ sub-populations. The positive CD8 or CD4 cells were gated for additional activation markers CD44 and CD62L and Treg markers CD25 and Foxp3 (for the CD4⁺ population). Proportions were calculated as percentages of total live cells in each gated box, according to the parent gate.

Borg D.J., Yap F.Y., Keshvari S., Simmons D.G., Gallo L.A., Fotheringham A.K., Zhuang A., Slattery R.M., Hasnain S.Z., Coughlan M.T., Kantharidis P, & Forbes J.M. (2017). Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction. Islets, 10(1), 10-24.

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Characterizing T Cell Adhesion and Chemokine Expression

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Whole blood and MLNs were used to analyze adhesion molecule- and chemokine receptor-expressing T cells. Whole blood and MLN cells obtained as described above were split into 2 vials with 100 μL in each aliquot, and these were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences) or Pacific blue-conjugated anti-CD8 antibodies (Biolegend). To investigate expressions of adhesion molecules and chemokine receptors, PE-conjugated anti-PSGL-1 antibodies (BD Biosciences), APC-conjugated anti-α4β7 integrin, FITC-conjugated anti-CD11a, and PerCP-Cy5.5-conjugated anti-CCR9 (Biolegend) were added. Stained cells were analyzed by five-color flow cytometry. Lymphocytes were gated on the basis of their forward- and side-scatter profiles. Fluorescence data were recorded, and results are presented as percentages of adhesion molecule- and chemokine receptor-expressing CD4⁺ and CD8⁺ lymphocytes. Representative flow cytometry plots are shown in Figure 2(c).

Hou Y.C., Wu J.M., Wang M.Y., Wu M.H., Chen K.Y., Yeh S.L, & Lin M.T. (2014). Glutamine Supplementation Attenuates Expressions of Adhesion Molecules and Chemokine Receptors on T Cells in a Murine Model of Acute Colitis. Mediators of Inflammation, 2014, 837107.

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Phenotypic Analysis of T Helper Cells

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To determine the phenotypes of Th cells, 100 μL aliquots of blood were incubated with a Pacific blue-conjugated anti-CD4 (BD Biosciences) Ab, fixed, and then permeated for intracellular cytokine staining. The following Abs were used for intracellular cytokine staining: Alexa Fluor 488-conjugated anti-interleukin- (IL-) 4 (Biolegend), APC-conjugated anti-interferon- (IFN-) γ (BD Biosciences), and PE-conjugated anti-IL-17A (Biolegend) Abs. To analyze Treg, 100 μL aliquots of blood were incubated with Pacific blue-conjugated anti-CD4 and APC-conjugated anti-CD25 (eBioscience) Abs. After incubation for 30 min, leukocytes were fixed and permeated with Foxp3 staining buffer (eBioscience). PE-conjugated anti-Foxp3 (Biolegend) Abs were then added for the intracellular staining of Foxp3.
After staining, red blood cells were lysed, and leukocytes were analyzed by flow cytometry. CD4-positive lymphocytes were gated on the basis of low forward and side scatter. Phenotypes of Th cells are presented as percentages of Th-associated cytokine-expressing cells in CD4-positive lymphocytes. Treg populations are presented as a percentage of CD25/Foxp3 double-positive cells in gated CD4-expressing lymphocytes.

Hou Y.C., Wu J.M., Wang M.Y., Wu M.H., Chen K.Y., Yeh S.L, & Lin M.T. (2015). Modulatory Effects of Astragalus Polysaccharides on T-Cell Polarization in Mice with Polymicrobial Sepsis. Mediators of Inflammation, 2015, 826319.

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Phenotyping of T Helper Cells

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To determine the phenotypes of Th cells, 100 μL aliquots of blood were incubated with Pacific blue-conjugated anti-CD4 (BD Biosciences), fixed and then permeated for intracellular cytokine staining. The following Abs were used for intracellular cytokine staining: Alexa Fluor 488-conjugated anti-IL-4 (Biolegend), APC-conjugated anti-interferon (IFN)-γ (BD Biosciences) and PE-conjugated anti-IL-17A (Biolegend) Abs. For analyzing the Treg subset, leukocytes were incubated with Pacific blue-conjugated anti-CD4 and APC-conjugated anti-CD25 (eBioscience) Abs. After incubation for 30 min, leukocytes were fixed and permeated with Foxp3 staining buffer (eBioscience). PE-conjugated anti-Foxp3 (Biolegend) Abs were then added to stain for intracellular Foxp3. After RBCs were lysed, the leukocytes were stained and analyzed by flow cytometry. CD4-positive lymphocytes were gated on the basis of low forward and side scatter. Phenotypes of Th cells are presented as percentages of Th-associated cytokine-expressing cells in CD4-positive lymphocytes. The Treg population is presented as a percentage of CD25/Foxp3 double-positive cells in gated CD4-expressing lymphocytes.

Shih J.M., Shih Y.M., Pai M.H., Hou Y.C., Yeh C.L, & Yeh S.L. (2016). Fish Oil-Based Fat Emulsion Reduces Acute Kidney Injury and Inflammatory Response in Antibiotic-Treated Polymicrobial Septic Mice. Nutrients, 8(3), 165.

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Immunophenotyping of T-Cell Subsets

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After washing in PBS containing 0·1% BSA, PBMCs were stained with Alexa-700-conjugated anti-CD3 (BD Biosciences Cat# 557943, RRID:AB_396952), Pacific Blue-conjugated anti-CD4 (BD Biosciences Cat# 558116, RRID:AB_397037), allophycocyanin (APC)-H7-conjugated anti-CD8 (BD Biosciences Cat# 560179, RRID:AB_1645481), PerCP-Cy5·5-conjugated anti-CD45RA (BD Biosciences Cat# 563429, RRID:AB_2738199), phycoerythrin (PE)-conjugated anti-CCR7 (BioLegend Cat# 353203, RRID:AB_10916391), FITC-conjugated anti-CD127 (Thermo Fisher Scientific Cat# 11-1278-42, RRID:AB_1907342), and PE-Cy7-conjugated anti-CD25 (Thermo Fisher Scientific Cat# A15857, RRID:AB_2534627). Then, cells were fixed and permeabilized using FOXP3 staining buffer set (Thermo Fisher Scientific Cat# 00-5523-00), according to the manufacturer’s instructions. Cells were then stained with APC-conjugated anti-FOXP3 (Thermo Fisher Scientific Cat# 17-4776-41, RRID:AB_1603281) and acquired on a FACS Aria III (BD Biosciences) running FACS Diva v8 software (Becton Dickinson). Data were analyzed using Kaluza v1·3 (Beckman Coulter).

Casas R., Dietrich F., Barcenilla H., Tavira B., Wahlberg J., Achenbach P, & Ludvigsson J. (2020). Glutamic Acid Decarboxylase Injection Into Lymph Nodes: Beta Cell Function and Immune Responses in Recent Onset Type 1 Diabetes Patients. Frontiers in Immunology, 11, 564921.

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