Anti cd4 pe
Anti-CD4-PE is a fluorescently-labeled antibody that binds to the CD4 surface marker on T lymphocytes. It is designed for use in flow cytometry applications to identify and quantify CD4-positive cells in biological samples.
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45 protocols using anti cd4 pe
Flow Cytometric Analysis of Immune Cells
Vitamin D Modulation of PBMC Phenotype
Cells were harvested 48 h posttreatments in polypropylene tubes, centrifuged at 700 × g for 5 min and washed with PBS. Extracellular staining was done with fluorochrome-labeled antibodies purchased from eBioscience (Santa Clara, CA, USA): fixable viability dye eFluor 506, anti-CD3 PE-Cy7, anti-CD4 PE, anti-CD8 eFluor® 450, anti-CD56 PE-Cy7, and anti-TIM-3 APC-Cy7. Samples were acquired on a BD-LSRFortessa™ flow cytometer, and data were analyzed in FACSDiva v.8.0.1 software. The gating strategies are shown in Figure
Basophil Activation Assay for Allergy
Signaling Pathways in Immune Cell Activation
Antibodies to IκBα, p-ERK, p-JNK, p-p38 (T180Y182), p-MKK3/6, p-MKK4, SYK, PLCγ1, Vav-1, SLP-76, PKCθ, cleaved caspase 3, caspase 9, and cleaved caspase 9 were purchased from Cell Signaling Technology; anti-p-p38 (Y323) was from ThermoFisher Scientific; anti-GAPDH Ab was from Chemicon; Abs to mouse CD3, CD28, and Zap70 were from Biolegend; anti-CD3-FITC, anti-CD4-PE, anti-NK 1.1-PE, and anti-CD69-PerCP Abs, and Annexin V-FITC apoptosis detection kit were from eBioscience; anti-Gadd45α Ab was from Santa Cruz Biotechnology.
Multicolor Flow Cytometry Panel
In vivo Immune Cell Labeling
Quantifying Regulatory T Cells in Mice
Cd244 Knockout Mice for Immunological Studies
Cd244-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).
Comprehensive Multiparametric Flow Cytometry
For ex vivo analysis, cells were stimulated with 25 ng/mL PMA (Sigma-Aldrich) and 1 g/mL ionomycin (Sigma-Aldrich) in the presence of 0.66 μL/mL Golgistop (BD PharMingen) for 6 h at 37 °C, 5 % CO2. Intracellular staining of IFN-γ and IL-4 was performed using Transcription Factor Staining Buffer Set (eBioscience). Data was collected by FACS Calibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR).
For cell quantization, blood sample (100 μL) was stained with the specific antibodies: anti-CD19-FITC, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD11c-APC, anti-F4⁄80-FITC, and anti-CD11b-APC. Erythrocytes were then lysed with Cal-lyse Lysing Solution (Invitrogen). After thoroughly mixing with 100 μL of Caltag Counting Beads (Invitrogen), 10,000 beads were acquired in the FACS Calibur flow cytometer for each sample.
Lymphocyte Proliferation and Phenotyping in Immunized Chickens
Lymphocytes isolated from different immune chickens were counted and diluted to 1 × 106 cells/mL. The cells of 500 μL were stained with 5 μL of anti-CD4-PE or 4 μL of anti-CD8-PE (ebioscience, San Diego, CA, USA). After 30 min, the cells were washed and examined by flow cytometry.
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