2.4. Flow Cytometry

TBCs express membrane proteins, e.g., taste specific receptors and ions channels, which have vital role in taste transduction. Flow cytometry-based analysis was performed using antibodies raised against the extracellular regions of these membrane proteins.

The flow cytometry protocol to identify and analyse cultured BTBCs was adapted from Menon et al. [15] and Sing et al. [16]. BTBCs were harvested using above stated enzymatic technique; and total cell number count and cell viability were determined. The BTBCs in suspension were centrifuged 400–800 g 4 °C for 5 min. The BTBCs cell pellet was re-suspended in ice cold wash buffer, i.e., MACS buffer, to an approximate cell density of 1 × 10⁷ cells/mL. MACS buffer was prepared using BSA (1%) and EDTA (2 mM) (Fisher Scientific) in PBS. After centrifugation, appropriate antibody concentration diluted in blocking buffer, i.e., 5% goat serum in PBS, was added and the cells were incubated for 15–30 min at 4 °C in the dark. The primary antibody was then removed and washed twice by centrifugation for 5 min using MACS buffer. All primary antibodies were prepared in three dilutions (i.e., 1:100, 1:200 and 1:400) unless specified. Appropriate fluorochrome-conjugated secondary antibodies, diluted in the blocking buffer, were added, and further incubated for 20–30 min at 4 °C in the dark. The BTBCs were washed twice with MACS buffer by centrifugation for 5 min. Cells were re-suspended in ice cold MACS buffer and cells were filtered through 85 µm mesh pluriStrainer filters prior flow cytometry analysis (pluriSelect Life Science, Leipzig, Germany). To identify and exclude dead cells, DAPI (0.02 μg/mL) was added just before flow cytometric analysis using MoFlo Astrios cell sorter (Beckman Coulter, High Wycombe, UK).

CD73 is membrane protein reported to be specifically expressed in Type III cells [17]. Therefore, flow cytometry analysis was performed using PE conjugated anti-CD73 (Abcam) to identify BTBCs that express CD73. Antibody titration and optimisation was performed using three different concentrations, i.e., 1:100, 1:200 and 1:400. If no positively stained cells were observed, the specificity and suitability of the antibody was further studied using immunocytochemistry technique and confocal microscopy.

TRPM5 is specifically expressed in Type II cells (TRCs), i.e., cells sensitive to bitter, sweet and umami taste. Therefore, BTBCs were labelled using rabbit anti-TRPM5 primary antibody (Invitrogen, Thermo Fisher Scientific, Paisley, United Kingdom) and anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen). To find optimum labelling conditions, the applications of both primary and secondary antibodies were titrated using three dilutions (i.e., 1:100, 1:200 and 1:400) and two different concentrations, respectively.

There are more than 25 taste receptors, i.e., T2Rs, responsible for bitter taste transduction. BTBCs were stained with primary antibody raised against the extracellular regions T2R7 as stated above. Cells were labelled using three dilutions (i.e., 1:100, 1:200 and 1:400) of mouse anti-T2R7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using and a secondary antibody goat anti-mouse Alexa Fluor 488 (Abcam, Cambridge, UK).