Fig. 2

a. Time kinetics of DiOC₆(3) uptake in NKE cells following treatment with Mannan. Mitochondrial Membrane potential mediated sequestration of DiOC₆(3) in mitochondria of NKE cells was assessed flowcytometrically as described in materials and method section. Briefly, cells were treated with mannan, and dye uptake with time was measured continuously using Ex λ 488 and Em λ 530. The results are representative of three independent experiments. Data was analyzed using FlowJo V10.1 software. Images to assess for changes in membrane potential using JC-1 dye (using green and red channels of ZOE™ Fluorescence microscope) were also acquired after 10 min (incubation with dye) as described in materials and method section. The acquired images were merged and analysed using inbuilt software provided with the microscope. b. Time kinetics of ROS generation in NKE cells after treatment with Mannan. ROS mediated oxidation of CM-H₂DCFDA and measurement of the fluorescent CM-DCF with time as an indicator of redox status of cells. Briefly, cells treated with mannan was harvested and thereafter oxidation of dye was measured flowcytometrically using Ex λ 488 and Em λ 530 as described under materials and methods. Obtained results presented are representative of three independent experiments. Data was analyzed using FlowJo V10.1 software. To confirm the changes in ROS levels following treatment of cells with mannan, the oxidation of CM-H₂ DCFDA dye was also measured using microplate reader, at Ex λ 488 nm and Em λ 530 nm. Results are representative of mean fluorescence and expressed as fold changes in mean fluorescence