Cellular immune response was assessed by counting the numbers of various types of functional T lymphocytes in vaccinated chickens. Two weeks after the boosting immunization and prior to challenge, five chickens were selected randomly from each group and peripheral blood samples were collected from the brachial wing vein using heparinized syringes. Peripheral blood mononuclear cells (PBMCs) were isolated and adjusted to 1 × 107 cells/ml. The samples (100 μl; 1 × 106 cells) were incubated for an hour at room temperature with mouse anti-chicken CD4-FITC, CD8-RPE and CD3-SPRD (Southern Biotech, Birmingham, AL, USA). Flow cytometry was performed on a FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA) and data were analyzed with CellQuest software (BD Bio-sciences).
Cd3 sprd
Manufactured by Southern Biotech
Sourced in United States
The CD3-SPRD is a laboratory instrument designed for the detection and analysis of CD3 proteins on the surface of T cells. It utilizes flow cytometry technology to provide precise quantification of CD3 expression levels. The core function of the CD3-SPRD is to facilitate the measurement and characterization of T cell immunophenotypes.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 rpe, by Southern Biotech (2 mentions)
Facscalibur, by BD (2 mentions)
Cd4 fitc, by Southern Biotech (2 mentions)
Cd8α fitc, by Southern Biotech (2 mentions)
Cd4 pe, by Southern Biotech (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Mouse anti chicken cd4 fitc, by Southern Biotech (1 mentions)
Cellquest software, by BD (1 mentions)
Cellquest, by BD (1 mentions)
Cell strainer, by BD (1 mentions)
Cd8a fitc, by Southern Biotech (1 mentions)
5 protocols using cd3 sprd
1
Quantifying T Cell Populations in Vaccinated Chickens
2
Splenic Lymphocyte CD4+/CD8+ Ratio Analysis
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The splenic lymphocytes were collected for the measurement of the CD4+/CD8+ ratio as previously described27 (link). Briefly, 1 × 106 splenic lymphocytes were incubated with anti-chicken CD3-SPRD, CD4-FITC and CD8-RPE (Southern Biotech, USA) at 4 °C for 30 min. Subsequently, lymphocytes were washed 3 times with phosphate buffer saline containing 1% fetal bovine serum, then resuspended and analyzed by FacsCalibur and CellQuest software (Becton Dickinson, USA). Viable lymphocytes were calculated based on light scatter (forward and side scatter) characteristics, and 10,000 events were analyzed for positive staining with SPRD, FITC and RPE antibodies.
3
Multiparametric Flow Cytometry Immunophenotyping
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Cells were diluted in staining buffer (PBS 1×, 10% FBS, 0.1% Sodium Azide) and 1 × 106 cells per well were transferred into 96 well-plates (V-shape), and washed twice with the same buffer. Staining was performed by resuspending the cellular pellet of each well with 100 μL of staining buffer including different combinations of antibodies, or as single-color stainings for compensation. Cells were incubated at 4 °C for 30 min and washed twice with staining buffer by centrifugation at 250 × g for 5 min.
Avian monoclonal antibodies (mAbs) (CD3-SPRD, CD4-FITC, CD8α-PE, CD8α-FITC and CD8β-PE) were purchased from Southern Biotech (Birmingham, AL, USA). Mononuclear cell suspensions from every thymus and spleen were stained with one and two mAb mix, respectively: CD3-SPRD, CD4-FITC and CD8α-PE in tube A (for thymocytes and splenocytes), and CD3-SPRD, CD8α-FITC and CD8β-PE in tube B (splenocytes). All antibodies were titrated in order to determine the optimal staining concentration of each one.
Positive cells were analyzed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and CellQuest software. Analysis was done on 20 000 events and discrete viable lymphoid cell populations were gated according to the forward/side scatter characteristics. Percentages of different lymphoid cell subpopulations in the thymus and spleen were determined by multiparametric analysis.
Avian monoclonal antibodies (mAbs) (CD3-SPRD, CD4-FITC, CD8α-PE, CD8α-FITC and CD8β-PE) were purchased from Southern Biotech (Birmingham, AL, USA). Mononuclear cell suspensions from every thymus and spleen were stained with one and two mAb mix, respectively: CD3-SPRD, CD4-FITC and CD8α-PE in tube A (for thymocytes and splenocytes), and CD3-SPRD, CD8α-FITC and CD8β-PE in tube B (splenocytes). All antibodies were titrated in order to determine the optimal staining concentration of each one.
Positive cells were analyzed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and CellQuest software. Analysis was done on 20 000 events and discrete viable lymphoid cell populations were gated according to the forward/side scatter characteristics. Percentages of different lymphoid cell subpopulations in the thymus and spleen were determined by multiparametric analysis.
4
Flow Cytometric Analysis of Bursal T-cells
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Lymphocytes were isolated from bursal samples and were used to study T-cell infiltration by flow cytometry as previously described (Carballeda et al., 2011 (link)). Briefly, bursas were mechanically disrupted in RPMI 1640 and cellular suspensions were passed through a 40 μm mesh (Cell Strainer, BD). Mononuclear cells were isolated by centrifugation over a Histopaque density gradient. About 1 × 106 cells per well were seeded on a 96-well plate and stained with different combinations of antibodies. Monoclonal antibodies (mAbs) (CD3-SPRD, CD4-PE, CD8α-FITC, Bu-PE) were purchased from SouthernBiotech (Birmingham, AL, United States). Cell suspensions were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States) and CellQuest software. The lymphocyte gate was defined by the forward/side scatter characteristics of the cells and 50,000 events were analyzed for each sample. Individual values of all experimental groups were normalized to the mean values of unchallenged healthy group.
5
Lymphocyte Analysis of Chicken Immunogenicity
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Lymphocytes were isolated from the BF as previously described [19] (link). Monoclonal antibodies (mAbs) (CD3-SPRD, CD4-PE, CD8a-FITC) from Southern Biotech (Birmingham, USA) were employed. Cell suspensions were analyzed with a BD FACSCalibur Flow Cytometer (BD Biosciences, USA) and CellQuest software. The lymphocyte gate was defined by the forward/side scatter characteristics of the cells and 30,000 events were analyzed. Six specific pathogen free chickens were used in each group. Inoculations were performed at 14 days post hatch (1st) and 28 days post hatch (2nd). Animals were challenged three weeks after the 2nd inoculation and euthanized 5 days post challenge.
The mean values of the bursae from three PBS-inoculated unchallenged SPF chickens were used for normalization of the values of all experimental groups.
The mean values of the bursae from three PBS-inoculated unchallenged SPF chickens were used for normalization of the values of all experimental groups.
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