inverted carl zeiss axiovert 200m motorized inverted fluorescence microscope  (Carl Zeiss)

Journal: Experimental eye research

Article Title: Positively charged amino acid residues in the extracellular loops A and C of lens Aquaporin 0 interact with the negative charges in the plasma membrane to facilitate cell-to-cell adhesion

doi: 10.1016/j.exer.2019.05.022

Figure Lengend Snippet: CTCA assay. (A). Over a monolayer of L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) corresponding cells loaded with CellTracker Red were plated. At the end of the procedure, the cells were imaged under an epifluorescent microscope. Cells/aggregates were counted and plotted. (B). Bar graph represents the number of dye-loaded L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) that remained attached to the matching cDNA construct-transfected L-cells (without dye) due to CTCA. Samples were tested using the fluorescence assay and incubated for 1h for CTCA. Compared to WT-AQP0, mutant AQP0 exhibited significantly low (P < 0.05) CTCA, denoted with a star for each sample. E-cadherin - positive control. (C). CTCA assay testing the possible mechanism of CTCA. The number of dye-loaded cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) that remained attached to untransfected L-cells, due to CTCA in the samples tested using the fluorescence assay with 1h of incubation are represented. Star on the mutants denotes a reduction in CTCA in comparison with WT AQP0. Note: Number of E-cadherin transfected cells that remained attached was much less than that of WT AQP0 transfected cells and represented with two stars.

Article Snippet: Using Zeiss Axiovert 200M motorized inverted fluorescence microscope, optimized Z-sectional digital images were acquired as described ( Kumari et al., 2013 ; Varadaraj et al., 2008 ).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Microscopy, Construct, Transfection, Fluorescence, Incubation, Positive Control, Comparison