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45 protocols using anti cd4 pe

1

Flow Cytometric Analysis of Immune Cells

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The population of each phenotype of immune cells in different groups was evaluated by using flow cytometric analysis as described previously [30 (link)]. In brief, splenocytes were stained with fluorescent antibodies, including anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-APC, anti-MHCII-FITC, anti-CD86-PE, anti-CD40-PE, anti-CD4-PE (eBiosciences, San Diego, USA), anti-IL-4-APC, anti-CD68-FITC, and anti-CD206-PE (BioLegend, San Diego, USA), according to the manufacturer’s instruction. The FlowJo software was used to analyze the percentages of various phenotypes of immune cells.
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2

Vitamin D Modulation of PBMC Phenotype

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In 96-well plates (BD, Franklin Lakes, NJ, USA), 100,000 viable PBMCs/well of 6 Colombian HCs were resuspended in 200 µL RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and were treated with calcidiol at 250 nM and calcitriol (active form of VitD) at 0.5 nM (both within the physiological range) or with 0.01% v/v EtOH as vehicle control.
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 S1 in Supplementary Material.
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3

Basophil Activation Assay for Allergy

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Whole blood samples from all control and experimental groups were collected via cheek puncture at D63 to conduct a basophil activation test (BAT) according to the method described by Torrero et al. [18 (link)]. Briefly, whole blood was incubated (1.5 h at 37°C) with RPMI-1640 medium (Lonza), αIgE (125 ng/ml, eBioscience, Breda, the Netherlands), or whey (20 μg/ml, DMV International) to activate basophils. After red blood cell lysis (Whole Blood Lysing Reagents, Beckman Coulter, Fullerton, CA, USA), cells were stained with anti-IgE-FITC, anti-CD49b-APC, anti-CD4-PE, and anti-B220-PE (eBioscience) to select the basophil population while excluding T and B cells. Median fluorescence intensity (MFI) of activation marker CD200R-PerCp-eFluor 710 was determined with flow cytometry using a FACS Canto II (BD Biosciences, Alphen a/d Rijn, the Netherlands).
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4

Signaling Pathways in Immune Cell Activation

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Concanavalin A, actinomycin D and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) were obtained from Sigma-Aldrich; mouse or human TNF-α was from PeproTech; α-galactosyl ceramide (α-GalCer; KRN7000) was from Enzo Life Science; R788 was from Cayman Chemical.
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.
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5

Multicolor Flow Cytometry Panel

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The following antibodies were purchased from eBioscience (San Diego, CA): anti-CD8a-FITC, anti-CD8a-PE, anti-CD4-FITC, anti-CD4-PE, anti-IFN-γ-PE, anti-IFN-γ-FITC, anti-TNF-α-APC, anti-PD-1 (CD279)-FITC, and anti-rat IgG-FITC. The anti-CD8a-APC-Cy7 and anti-PD-1 (CD279)-PE antibodies were purchased from BioLegend (San Diego, CA) and the anti-CD127-Alexa fluor 647 antibody was from Bio-Rad Laboratories (Hercules, CA). The annexin V-FITC apoptosis detection kit was purchased from BioVision, Inc. (Milpitas, CA).
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6

In vivo Immune Cell Labeling

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For in vivo labeling of circulating immune cells, anti-CD4-PE (eBioscience, RM4-4, 1:400) and anti-CD8β-PE (eBioscience, 1:400) were diluted in PBS and administered by IV injection 5 minutes before harvest(Anderson et al., 2014 (link)) Alternatively, anti-CD45-PE-CF594 (30-F11, BD Biosciences, 1:200) or anti-CD45-APC-ef780 (30-F11, eBioscience 1:40) were also used for intravascular labeling and were administered 2 or 5 minutes, respectively, before sacrifice. Mice were exposed ad libitum to drinking water containing dissolved FTY720 at a concentration of 2 μg/mL for 2 weeks starting at 6 weeks post-initiation.
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7

Quantifying Regulatory T Cells in Mice

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Lymph node and spleen cells from mice were harvested after treatments. Cells were isolated and stained using surface markers (anti-CD4-PE, eBioscience) and then an intracellular marker (anti-Foxp3-APC, eBioscience) using intracellular fixation/permeabilization kit (eBioscience, San Diego, CA). And CD4+Foxp3 Tregs were analyzed using a FACSCalibur (BD, Biosciences).
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8

Cd244 Knockout Mice for Immunological Studies

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C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
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).
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9

Comprehensive Multiparametric Flow Cytometry

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Cell surface markers were stained with these specific antibodies: anti-CD19-PE, anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC, 7-AAD, and anti-annexin-V-FITC (eBioscience); anti-CD19-APC, anti-B220-Per-cy5.5, anti-IgM-FITC, anti-AA4.1-PE, anti-CD23-eFluor647, and anti-CD8-APC (Biolegend).
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.
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10

Lymphocyte Proliferation and Phenotyping in Immunized Chickens

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Lymphocytes were isolated from the peripheral blood of immunized chickens using commercial kits (Solarbio, Beijing, China) according to the manufacturer’s instructions. Then, the lymphocytes were seeded and cultured in a culture medium with 20 μL of ConA. The plates were cultured in an incubator with 5% CO2 for 44 h. The proliferation ratio was determined by the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). The cells were treated with 10 μL of CCK-8 solution and incubated at 37 °C for 4 h. The absorbance was measured by using an automated microplate reader (Bio-Rad, Japan) at 450 nm, and the stimulation index (SI) was calculated using the equation as follows: SI=OD450 of stimulated cellsOD450 of unstimulated cells ×100%.
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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