Cecal tonsil samples were collected from one bird/pen (n = 6) at d14 and d21 of age. For flow cytometry analysis, both treatment groups consisted of six samples, in duplicates, for each time point. Cecal tonsil samples were teased over a 0.4 μm cell strainer (Sigma, MO) with 2 mL RPMI-1640 media to obtain a single-cell suspension. Single-cell suspensions were concentrated for lymphocytes by density centrifugation over Histopaque (1.077 g/mL; Sigma, MO). For CD4+/CD8+ analysis, single-cell suspensions of the cecal tonsils (1 × 106 cells) were incubated with FITC-conjugated mouse anti-chicken CD4, PE-conjugated mouse anti-chicken CD8 (Southern Biotech, Birmingham, AL) at 1:200 dilution, and unlabeled mouse IgG at 1:200 dilution in a 96-well plate for 20 minutes. After incubation, cells were washed twice to remove unbound primary antibodies at 400 ×g for 5 minutes using wash buffer (1× PBS, 2 mM EDTA, 1.5% FBS). After washing, cells were analyzed using cytosoft software (Guava Easycyte, Millipore, Billerica, MA). The CD4+ and CD8+ and CD4+/CD8+ cell percentages were analyzed after gating cells based on forward-scatter and side-scatter plot for lymphocytes.
Fitc conjugated mouse anti chicken cd4
Manufactured by Southern Biotech
Sourced in United States
FITC-conjugated mouse anti-chicken CD4 is a laboratory reagent used to detect and quantify CD4-positive cells in chicken samples. It consists of a fluorescein isothiocyanate (FITC) dye conjugated to a monoclonal antibody specific for the chicken CD4 surface protein.
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Lab products found in correlation
Pe conjugated mouse anti chicken cd8, by Southern Biotech (2 mentions)
Apc conjugated mouse anti chicken cd3, by Southern Biotech (2 mentions)
Pe conjugated mouse anti chicken cd8α monoclonal antibodies, by Southern Biotech (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Histopaque, by Merck Group (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
5 protocols using fitc conjugated mouse anti chicken cd4
1
Cecal Tonsil Lymphocyte Populations
2
Chicken Lymphocyte Subset Analysis
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3 × 105 cells of PBL or tissue single-cell suspensions were simultaneously incubated with APC-conjugated mouse anti-chicken CD3+, FITC-conjugated mouse anti-chicken CD4+, and PE-conjugated mouse anti-chicken CD8α+ monoclonal antibodies (SouthernBiotech, Birmingham, USA) in the dark at 4 °C for 30 min. After three washes with PBS, the labeled cells were analyzed by flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA) within 12 h. The data were analyzed by the software of FlowJo V10 (TreestarInc, Ashland, OR, USA). For the phenotype identification of the CD8+ T cell, FITC-conjugated mouse anti-chicken CD8β+ and APC-conjugated mouse anti-chicken CD4+ monoclonal antibodies (SouthernBiotech, Birmingham, USA) were also used.
3
Simultaneous Multicolor Flow Cytometry
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The 3 × 105 cells of PBL were simultaneously incubated with APC-conjugated mouse anti-chicken CD3+, FITC-conjugated mouse anti-chicken CD4+, and PE-conjugated mouse anti-chicken CD8α+ monoclonal antibodies (SouthernBiotech, Birmingham, AL) in the dark at 4°C for 30 min. After 3 washes with PBS, the labeled cells were analyzed by flow cytometer (CytoFLEX; Beckman Coulter, Brea, CA) within 12 h. The data were analyzed by the software of FlowJo, V10 (TreestarInc, Ashland, OR).
4
Arginine's Effect on Immune Cells
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On d 21, 28, and 35 posthatch, one cecal tonsil and spleen were collected in RPMI-1640 medium from one bird per pen to perform flow cytometry to determine the effect of 125% arginine on the CD4+ T-cells, CD8+ T-cell ratio (Mortada et al., 2021 ) as previously described. Briefly, the cecal tonsils and spleen were collected in 2 mL and 5 mL of RPMI-1,640 media, respectively and single-cell suspensions of cecal tonsil and spleen lymphocytes were obtained. The single cell suspension containing 1 × 106 cells/mL (15 µL) was incubated with FITC-conjugated mouse anti-chicken CD4 at 1:250 dilution, PE-conjugated mouse anti-chicken CD8 (Southern Biotech, Birmingham, AL) at 1:450 dilution, and unlabeled mouse IgG at 1:100 dilution in a 96 well plate for 20 min. After incubation, the plates were washed with a wash buffer and the CD4+ and CD8+ T-cell percentages were analyzed using Cytosoft software (Guava EasyCyte, Millipore, Billerica, MA) after gating, based on forward scatter and side scatter plots for lymphocytes.
5
Flow Cytometry Analysis of Chicken Immune Cells
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The cells isolated from the jejunum were stained for flow cytometry. The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Thermo Fisher Scientific, Seoul, Republic of Korea) was used to analyze the live cells. The stain was diluted 1:1000 in PBS and stained with other antibodies. Macrophages and B cells were stained with PE-conjugated mouse anti-chicken Mono/Macro (8420-09, Southern Biotech, Birmingham, AL, USA), FITC-conjugated mouse anti-chicken MHCII (8350-02, Southern Biotech, Birmingham, AL, USA), and Alexa Fluor 647-conjugated mouse anti-chicken Bu-1 antibodies (8395-31, Southern Biotech). T cell subsets were stained with Pacific Blue-conjugated mouse anti-chicken CD3 (8200-26, Southern Biotech, Birmingham, AL, USA), FITC-conjugated mouse anti-chicken CD4 (8210-02, Southern Biotech, Birmingham, AL, USA), SPRD-conjugated mouse anti-chicken CD8α (8220-13, Southern Biotech, Birmingham, AL, USA), and PE-conjugated mouse anti-chicken TCRγδ antibodies (8230-09, Southern Biotech, Birmingham, AL, USA). Each antibody was diluted 1:200 in PBS and used for staining. The cells were measured using an FACS Canto II (Becton Dickinson, Heidelberg, Germany) and analyzed using FlowJo software v10.7.1 (Tree Star Inc., Ashland, OR, USA).
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