Chicken splenic T-cell subsets were detected as previously described [16 ]. Following filtration using a 300-mesh nylon gauze, cell suspension was washed twice using prechilled PBS, centrifuged for 5 min at 200 g, and the resultant supernatant was discarded. Pellets were resuspended in PBS, and 100 μL of suspension was transferred to a new culture tube. Cells were incubated with 10 μL mouse antichicken CD4-FITC and CD8-PE (SouthernBiotech, Birmingham, AL, USA), respectively. Following incubation at 25°C for 30 min, 2 mL PBS was added and centrifuged for 5 min at 200 g, and supernatant was discarded. Cells were resuspended in 400 μL of 1 × binding buffer (BD Pharmingen) and measured by the use of a flow cytometer (BD Bioscience).
Cd8 pe
Manufactured by Southern Biotech
Sourced in United States
CD8-PE is a fluorescently labeled antibody that binds to the CD8 cell surface antigen. It is commonly used in flow cytometry applications to identify and quantify CD8-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 apc, by Southern Biotech (2 mentions)
Mouse anti chicken cd4 fitc, by Southern Biotech (1 mentions)
Binding buffer, by BD (1 mentions)
Anti chicken cd3 fitc, by Southern Biotech (1 mentions)
Fortessa facs machine, by BD (1 mentions)
Igg1 pe, by Southern Biotech (1 mentions)
Igg1 fitc, by Southern Biotech (1 mentions)
Amnis flowsight imaging flow cytometer, by Merck Group (1 mentions)
Cd4 fitc, by Southern Biotech (1 mentions)
4 protocols using cd8 pe
1
Chicken Splenic T-Cell Subset Analysis
2
Splenic Lymphocyte Characterization in Chickens
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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.
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
Broiler's Intestinal Immune Profiling
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The cellular mucosal immune system status of the broiler's intestinal tract, intraepithelial lymphocytes (CD3+ T lymphocytes, CD3+CD8+ cells, CD3+CD4+ cells, and CD3+CD4+CD8 cells) were measured. At post mortem, intraepithelial T lymphocytes were estimated from isolated jejunum of 40 birds (1 bird per cage) at 42 d of age according to the method defined by Röhe (2014) . The intestinal cell suspensions were stained with either a cocktail of T lymphocyte CD marker antibodies (CD3-AF647, CD4-FITC and CD8-PE; Southern Biotech, Birmingham, AL, USA) or a cocktail of isotype control antibodies (IgG1-PE, IgG1-FITC, and IgG1-AF647; Southern Biotech, Birmingham, AL, USA) for 30 min on ice in the dark according to the manufacturer's instructions.
Flow cytometric data were acquired on an Amnis FlowSight imaging flow cytometer (Millipore,Burlington, Massachusetts , USA). CD4-FITC and CD8-PE were detected using the 488 nm laser, and the CD3-AF647 was detected using the 633 nm laser. Signals from the isotype antibody cocktail were subtracted from the T lymphocyte CD antibody fluorescence. From the total cell population that was acquired, the CD3+ intact cell population was gated. Sub-gates were applied to the intact CD3+ cells population to determine the proportion of cytotoxic T lymphocytes (CD3+CD8+), T helper lymphocyte (CD3+CD4+) and double-stained T lymphocytes (CD4+CD8+).
Flow cytometric data were acquired on an Amnis FlowSight imaging flow cytometer (Millipore,
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