Facs lsr fortessa

The ability of PMVDS to deliver encapsulated antigen to dendritic cells was assessed using an in-vitro system with DC2.4 cells. The antigen (ova) was labeled with FITC to facilitate quantification of the antigen delivery. The cells were incubated with 25 μg of FITC-ova as a solution in 50 mM phosphate buffered saline, pH 7.4 (PBS), encapsulated inside InAc particles (PMVDS) or PLGA particles as a delivery system. Control cells were treated with PBS without FITC-ova. After 1 h of incubation at 37 °C and 5% CO₂, the cells were washed thrice with PBS, trypsinized, and fixed in 4% w/v paraformaldehyde for 5 min. The proportion of cells receiving FITC-ova and the amount of FITC-ova received per cell was quantified using flow cytometer (BD Biosciences FACS LSR Fortessa and analyzed with FlowJo™). Similarly, to study the persistence of antigen inside the dendritic cells, DC 2.4 cells were cultured on glass coverslips and treated as mentioned above. After 1 h of treatment, the cells were washed thrice to remove FITC-ova or particles, which are not associated with the cells. Subsequently, the cells were incubated in a medium devoid of the FITC-ova. At different time points, (1 h, 12 h, and 24 h) the cells were washed, fixed with 4% w/v paraformaldehyde, and the antigen levels inside the cells were observed under a fluorescence microscope [15 ,18 (link)].