Zoe fluorescent imager

Time kinetics of DiOC₆(3) uptake as indicator of alterations in membrane potential in NKE cells after treatment with mannan (20 μg/ml) was determined flowcytometrically (BD LSR II flow cytometer, BD Biosciences, USA). Autofluorescence from each cell suspension was acquired for 1 min and thereafter DiOC₆(3) was added (5 nM final concentration) to determine dye uptake for 10 min at Ex λ 488 nm and Em λ 530 nm. The acquired data was analyzed using FlowJo V10.1 software and is representative of findings from three independent experiments.
To confirm the alterations in MMP in following various treatments NKE cells were stained with 100 nM JC-1 (final concentration) dye for 10 min (to correlate MMP changes with cyanine dye used for flowcytometry) and image were acquired using ZOE™ Fluorescent imager (Bio-Rad, USA) using both red and green channels LEDs. Images were merged using inbuilt software provided along with microscope.

Comet assay was performed as given by Olive and Banáth (2006) (link), with slight modification. Cell pellets containing 5,000 cells were suspended in 0.8% low melting agarose in 0.9% saline solution, mixed well, and poured onto a frosted slide, and the coverslip was placed over the gel. After solidification, the slides were incubated overnight in freshly prepared lysis solution (2.5 M NaCl, 100 Mm Na. EDTA, 1% Triton-X 100, 10% DMSO) at 4°C. The slides were washed with freshly prepared alkaline buffer (300 Mm NaOH, 1 Mm NaEDTA, PH.13), followed by electrophoresis at 24 V for 30 min. The slides were washed with 0.4 M Tris-base and stained with EtBr solution. Imaging was carried out using ZOE fluorescent imager (Bio-Rad).

A live dead viability/cytotoxicity kit (Invitrogen Cat# L3224) was used to assess hydrogel cytotoxicity. Prepared hydrogels remained protected from light at 4°C for up to 16 h before cytotoxicity testing. Prior to introduction to the 70–80% confluent monolayer, each hydrogel was soaked in 70% ethanol for 10 min protected from light at room temperature, soaked in 3 mL culture media for 5–10 min twice, then gently placed into a 12 well dish. Bright field images were taken at 48 h of co-culture with human neonatal foreskin fibroblast cells (ATCC CRL-2097), then hydrogels were gently removed from the 12 well plates. Culture media was carefully discarded without disturbing the monolayer. Cells were washed three times using 1 mL of dye solution (4 μM Ethidium homodimer-1 and 0.4 μM Calcein AM dye from the kit in D-PBS). After the wash, the dye solution was removed and discarded. A final application of 500 μL dye solution was added and the plate was incubated at room temperature for 1 h, then wrapped in foil and protected from light until imaging on the Bio-Rad ZOE Fluorescent Imager.