To obtain splenic lymphocytes, five chickens from each group were respectively euthanized 1 week after the first immunization (week 3) and 1 week after booster immunization (week 4). The lymphocytes were then separated as described in 2.9. To analyze the percentages of CD4+ T lymphocyte subsets, obtained splenic lymphocytes (106 cells suspended in 100 μl PBS) were dually stained with anti-chicken CD3-FITC (Southern Biotech, Birmingham, AL, USA) and CD4-APC (Southern Biotech) for 30 min at 4°C in the dark. As for the proportions of CD8+ T-lymphocyte subsets, 106 splenic lymphocytes suspended in 100 μl PBS were dually stained with anti-chicken CD3-FITC and CD8-PE (Southern Biotech). After being washed thrice in PBS, lymphocytes were characterized by flow cytometry (Beckman Coulter Inc., Brea, CA, USA). Before cell sorting, fluorescence compensation was conducted using the fluorescence minus one (FMO) control. Each group consisted of five replications, and each replication was measured once.
Cd4 apc
Manufactured by Southern Biotech
Sourced in United States, United Kingdom
CD4-APC is a fluorescently conjugated antibody that binds to the CD4 surface receptor on T helper cells. It is used in flow cytometry applications to identify and quantify CD4-positive cells.
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Lab products found in correlation
Cd8 pe, by Southern Biotech (2 mentions)
Anti chicken cd3 fitc, by Southern Biotech (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Gata3 pe, by BD (1 mentions)
Il 10 pe, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Fortessa facs machine, by BD (1 mentions)
Facsort flow cytometer, by BD (1 mentions)
Cd3 pe, by Southern Biotech (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
4 protocols using cd4 apc
1
Splenic Lymphocyte Characterization in Chickens
2
Comprehensive Mucosal Immune Profiling
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For surface marker staining, ileal LPLs were stained with CD4 APC (SouthernBiotech, F2100403, AL), CD25 APC (BIO-RAD, MEM-181, CA), CD11b PE-Cy7 (SouthernBiotech, 8420, AL), CD19 Pacific blue (SouthernBiotech, 8395, AL), and CD11c FITC (Invitrogen, 25-0116-42, CA) antibodies. For analysis of intracellular signaling in LPLs, cells were fixed with a Fixation/Permeabilization Solution Kit (BD Pharmingen, CA) and stained with RORγt FITC (BD Pharmingen, 563621, CA) and GATA-3 PE (BD Pharmingen, 560074, CA) antibody. For intracellular cytokine staining, LPLs were stimulated with PMA (50 ng/mL), and Ionomycin (1,000 ng/mL) in the presence of Brefeldin A (5 μg/mL) for 6 h. Then cells were fixed, permeabilized and subsequently stained with IL-10 PE (Biolegend, 505007, CA) and TNF-α FITC (Bioss, bs-10802R-AF488, Beijing, China) antibody. Samples were detected with a BD FACSCanto II (BD Pharmingen, CA) and analysed with FlowJo software, with isotype or unstained controls to determine gating.
3
Immunophenotyping of Lymphocytes
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Lymphocytes were extracted from thymus glands as described above and single cells were resuspended in 1x PBS +1% FBS (buffer) in 3 ml tubes. 0.1 μg of CD4-APC, 0.1 μg of CD8-PE and 0.5 μg CD3-FITC (SouthernBiotech, Birmingham, AL: anti-CD4-APC (cat# 8210–11, lot#: C1706-R846X); anti-CD8A-PE (cat# 8220–09; lot #: L2413Y276B); anti-CD3-FITC (cat # 8200–02, lot #: E3813-QL26B) were added to 100 μl of the lymphocyte solution containing 1 x 106 lymphocytes and incubated for ½ hr in ice in the dark. Preliminary titration experiments were conducted to assess antibody requirements for optimal detection of the cell populations. After incubation, buffer was added to the top of the tubes containing the cell solutions. Cells were centrifuged, buffer was aspirated and 100 μl of fresh buffer added. Cell samples were filtered using BD Falcon Cell-Strainer Cap tubes and analysed in the Flow Cytometry Core Facility at Weill Cornell Medical College using a BD Fortessa FACS machine. Data were analysed using FlowJo software (FlowJo LLC, Becton Dickinson).
4
Isolation and Characterization of PBMC
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At 21 d, peripheral blood mononuclear cell (PBMC ) was isolated from peripheral blood samples using Ficoll density centrifugation according to the method of Tan et al. (2015) (link). Briefly, wing venous blood was collected and layered on top of the separation medium (Histopaque 1077; Sigma Chemical Company, Boston, MA) in tubes (1:1) and centrifuged at 200 g for 30 min (25°C). Then the PBMC at the plasma–Ficoll interface were collected. Cold RPMI-1640 medium (containing 5.0% inactivated fetal bovine serum, 100 U (0.0599 mg) penicillin/mL, 100 mg streptomycin/mL, and 10 mM-HEPES) was added to the tube containing the PBMC. After that, the cells were washed 3 times with cold RPMI-1640 medium by centrifugation at 100 g for 10 min (4°C). Then the samples were measured and analyzed using a FACSort flow cytometer (Becton Dickinson, Mountain View, CA) and Cell Quest software. First, the PBMC were incubated with specific antileucocyte monoclonal antibodies. The monoclonal antibodies (CD3-PE, CD4-APC, CD8-FITC) were purchased from Southern Biotech (Birmingham, AL). The cells were incubated with antibody at 4°C for 30 min then washed 3 times with PBS. Then samples were analyzed using the flow cytometer. Lymphocyte and monocyte subpopulations were gated based on forward and side-scatter characteristics, and the PBMC subpopulation counts were calculated based on 100 gated lymphocytes.
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