Monoclonal antibodies for flow cytometry (mouse anti-porcine CD4-PE and CD8a-FITC) were produced by Southern Biotech (Birmingham, AL, USA). A total of 100 μL of porcine blood was mixed with 50 μL saline and incubated with 2 μL of anti-CD4 and 2 μL of anti-CD8a for 30 min in the dark at ambient temperature. Following incubation with 600 μL (10% v/v) lysing solution (BD, Franklin Lakes, NJ, USA) for 10 min, the remaining cells were washed twice in PBS, centrifuged at 500g for 5min, resuspended in a total volume of 300 μL PBS, and assayed in a FACScan flow cytometer (BD, USA). First electronic gate was set on lymphocytes and then gated CD4+ T cells in Y axis and CD8+ T cells in X axis as Figure 1 . Dead cells were excluded according to the forward scatter and side scatter.
Cd8a fitc
Manufactured by Southern Biotech
Sourced in United States
CD8a-FITC is a fluorescently-labeled antibody that binds to the CD8a surface marker. It is a tool used for the identification and analysis of CD8+ T cells in flow cytometry applications.
Automatically generated - may contain errors
2 protocols using cd8a fitc
1
Porcine T-cell Enumeration by Flow Cytometry
2
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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