FACS Based Analysis
Flow cytometry has various research applications including immunophenotyping, measuring intracellular cytokine production, cellular proliferation, assessing cell viability and analysis of cell cycle, rare events, stem cells and fluorescent proteins.
Neutrophils were isolated from fresh peripheral blood using a commercially available isolation kit (details) and the purity of cells was subsequently analysed by FACS for neutrophil surface markers (CD45 and CD66b). Figures A 1-4 represent the gating strategy employed for characterising the neutrophils. The neutrophil population was subsequently stimulated with [concentration] TNFα for 4 hours and an increase in cells expressing the LFA-1 surface antigen was observed (B1, 2).
T-cell subpopulation characterisation: CD4+, CD27+, CD28+
T-cell lymphocytes were isolated from peripheral blood using commercially available isolation kits. To characterise the subpopulation of CD4+T-cells, the lymphocytes were stained for surface markers (CD4, CD27, CD28) along with viability dye and analysed by FACS. The CD4+T cells were further sub-characterised and 82.13% cells were positive for CD4, CD27 and CD28. The expression of these co-stimulatory markers are helpful in investigating the immune responses in young and old donors.
Regulatory T (TReg)-Cell Characterisation: CD4+CD25+CD127dim/-
Regulatory T cells (T Reg) were isolated from peripheral blood using commercially available isolation kits.
The cells were labelled with CD4, CD25 and CD127. It was observed that 45.57% of the cells were CD4+CD25+; within this a sub-population of 92.20% of the CD4+CD25+ cells expressed low levels of the CD127 surface antigen.
THP-1 characterisation after PMA-differentiation
Stimulation of THP-1 cells (human monocyte model) with PMA induces differentiation and expression of a macrophage-like phenotype. The PMA-differentiated THP-1 cells were stained with macrophage surface markers (CD11; CD40) and analysed using FACS. Figures A and B demonstrate that the expression of CD11 and CD40 was higher following PMA-induced differentiation of THP-1 cells (red) when compared to undifferentiated cells (green).
Differentiation of primary monocytes to M0, M1 and M2 MΦ
CD14+ monocytes were isolated from PBMCs using a commercially available kit. Monocyte differentiation to M0 macrophages (Panel A) was induced and inhibited by M-CSF and SB203580, respectively. Polarisation of M0 macrophages to M1 macrophages (Panel B) was induced by M1 cytokine cocktail and inhibited following treatment with Dexamethasone. Polarisation of M0 macrophages to M2 macrophages (Panel C) was induced by M2 cytokine cocktail and inhibited following treatment with Tofacitinib. Characterisation of M0, M1 and M2 macrophages under experimental conditions was performed by flow cytometric analysis of the expression of cell suface markers (CD86, CD80 and CD206).
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