Staining of colonocytes - surface Antigens and flowcytometery for IgA, IgG, toll-like receptor 2 and 4

The isolated Colonocytes from stool sample (approximately 25,000–50,000 cells) were suspended in 1 ml of PBS and washed with 2 ml of 1% BSA in PBS. The cell suspension was centrifuged at 2,000 rpm for 5 min. at 4°C and supernatant was discarded. The washing was repeated with PBS. The cell pellet was suspended in PBS and 100 μl aliquot was prepared in the running tube. Processing of sample for flowcytometry was performed as per standardized lab protocol. To measure the TLRs, IgA, and IgG receptor concentration on viable colonocytes, TLR2-APC conjugated, TLR4-PE-Cy7 conjugated, IgA-FITC conjugated, and IgG-PE conjugated antibodies were used and at least 5 lakhs events were acquired in BD-FACS Canto, 6 color (BD Biosciences, San Jose, USA). Data analysis was performed by BD-FACS-Diva software. Dot plot representation was shown in Figure 2.

Dot plots of flow cytometry demonstrating the expressions of IgA, IgG, TLR2, and TLR4. Relative expressions of markers were analyzed on the basis of their pattern in unstained tube. Figure a and a1): Forward scatter (FSC) and side scatter (SSC) pattern of the cells. Figure b, b1; c, c1; d, d1 and e, e1): Unstained and stained tubes for IgA, IgG, TLR2, and TLR4 expression, respectively