biotinylated mouse anti guinea pig cd8 antibody  (Bio-Rad)

Journal: Scientific reports

Article Title: Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge.

doi: 10.1038/s41598-023-45898-3

Figure Lengend Snippet: Figure 1. BCG-vaccination elicits CD1b-restricted, PIM6-specific T cell-responses. Four weeks after BCG-vaccination PBMCs were isolated. Autologous, CD1b-expressing APCs and non-adherent lymphocytes were stained with CFSE and stimulated with mycobacterial antigens. CFSE dilution was analyzed by flow-cytometry after five days. (A) Seven guinea pigs were control-treated. Eight guinea pigs were BCG-vaccinated. After four weeks, T cell-proliferation in response to the indicated mycobacterial antigen was assessed. The graphs on the left depict the gating strategy. In the diagrams, black bars represent the group mean as determined by nested-data-analysis. Error bars represent the standard error of the mean. Asterisks indicate the level of significance as determined by nested-t-test in comparison to the medium control. (B) CFSE-low, PIM6-reactive lymphocytes were stained for a general T cell-marker, CD4 and CD8. The small panel at the left shows the gating strategy. The larger pseudo-color graph representatively shows the CD4- and CD8-distribution. The dot blot on the right shows the relative distribution for T cell-markers in PIM6-reactive cells four weeks after BCG-vaccination. Red symbols represent the normal T cell- and CD4-CD8-distribution in PBMCs from naïve control guinea pigs. Asterisks indicate the level of significance as determined by unpaired t-test. (C) Lymphocytes were stimulated with PIM6 as described above. After 24 h RNA was harvested. Cytokine-transcript levels were determined by qRT-PCR in relation to β-Actin. Open bars represent non-stimulated controls, black bars represent transcript levels after PIM6-stimulation. The left panel shows the results for BCG-vaccinated animals, the right panel shows the upregulation of IFNγ, GM-CSF and IL17 for naïve, non-immunized animals. Black bars represent the group mean. Error bars indicate the standard error of the mean. Asterisks depict the level of significance as determined by Wilcoxon-matched-pair-signed-ranked-test in comparison to non-stimulated medium controls. (D) IFNγ-transcript levels were assessed in the presence of CD1b-blocking or isotype-matched control antibodies. Individually colored circles represent transcript levels of eight BCG-vaccinated guinea pigs four weeks after vaccination. Asterisks depict the level of significance as determined by Wilcoxon-matched-pair-signed-ranked-test as indicated.

Article Snippet: CFSE-negative lymphocytes were further characterized by a triple-staining using allophycocyanin-conjugated mouse-anti-guinea-pig-T cell-antibody (AbD Serotec, Germany); phycoerythrin-conjugated mouse-anti-guineapig-CD4 antibody (AbD Serotec) and biotinylated mouse-anti-guinea-pig-CD8 antibody (kindly provided by Dr. Hubert Schäfer, Robert Koch-Institut, Germany).

Techniques: Isolation, Expressing, Staining, Flow Cytometry, Control, Comparison, Marker, Dot Blot, Quantitative RT-PCR, Blocking Assay