The optimisation of labeling methods for the tracking of BCG antigen into the exosome pathway
Background – Exosomes are small vesicles released from all cell types. Antigens in the
exosomes of infected cells are able to sensitize immune response. Advanced technology of
microscopy and fluorescence facilitate antigens tracking in exosomes and optimisation of dyes
has to be conducted.
Methods – BCG strain FO was cultured and labeled with two different dyes: PKH and FITC. The
labeling was assessed using confocal microscopy and FACS. Exosome isolation of uninfected
THP-1 cells and BCG-infected THP-1 cells was then performed. The mean difference of size
and concentration of both groups were then analysed using Mann-Whitney test.
Results – The images brightness of labeled BCG are the same in the FITC concentration of 50,
100, and 500 ug/mL. The mean size of exosomes of control and treated groups are 127.5 nm
and 112.4 nm, respectively (p=0.4857). The exosome concentration of BCG-infected THP-1
cells is 0.5375 x 1011 particles/ml and the one of the control is 1.444 x 1011 and particles/ml
THP-1 cells (p=0.0286).
Conclusion – The brightness of the BCG labeling does not always increase along the given
FITC concentration. The size of macrophage-secreted exosomes is a little higher than the range
of normal exosome, and exosomes concentration of infected cells is not necessarily higher than