Flow Cytometry (FACS)
Flow cytometry is a laser-based technology used to analyse the characteristics of cells or particles. It is a widely used method for analysing the expression of cell surface and intracellular molecules, characterising and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analysing cell size and volume. It allows simultaneous multi-parameter analysis of single cells.
Neutrophil characterisation
Neutrophils were isolated from fresh peripheral blood using commercially available isolation kit and the purity of cells analysed by FACS for neutrophil surface markers (CD45 and CD66b). Data represents the gating used for characterising the neutrophils.
The neutrophil population was stimulated with TNF-alpha for 4 hours. A difference in the LFA-1 surface antigen expression between unstimulated (B.1) and stimulated (B.2) neutrophils was observed.
THP-1 characterisation after PMA-differentiation
THP-1 (a model for human monocytes cells) on stimulation with PMA, differentiate into mature macrophage-like phenotype. The PMA-differentiated THP-1 cells were stained with macrophage surface markers (CD11; CD40) and analysed using FACS. Expression of CD11 and CD40 was higher in PMA-differentiated THP-1 cells (red) when compared to undifferentiated THP-1 (green) cells.
T lymphocytes were isolated from fresh peripheral blood. To characterise sub-population of CD4+ T cells, lymphocytes were stained for surface markers (CD4, CD27, CD28) along with viability dye and analysed by FACS. CD4+ T cells were further sub-characterised and 82.13% of cells were observed to be positive for CD4, CD27 and CD28 surface markers. Expression of these co-stimulatory markers are helpful in investigating their immune responses.
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