Plan neofluar 20x 0.50 ph2 objective

Cuticle sensitivity to hypochlorite was tested as described^{24 (link), 29 (link)} with modifications. In brief, individual animals (L4 larvae or young gravid adults) were transferred into a 10 μl drop of freshly prepared 2%(v/v) alkaline hypochlorite solution (4 ml 5% sodium hypochlorite, 2.5 ml 4 N NaOH, 3.5 ml dH₂O) on parafilm and the time taken until first visible rupture was noted.
Cuticle permeability to acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) was assayed as described^{19 , 26 (link), 31 (link), 34 (link)} with modifications. L4 larvae were washed from plates with M9 buffer prior to staining with AO or DAPI (5 μg/ml each in M9 buffer) for 15 minutes at room temperature with gentle agitation. Subsequently, worms were washed three times with M9 buffer, followed by fluorescence imaging. For microscopy, worms were mounted onto 3%(w/v) agarose pads, anaesthetised with 10 μl of 1 mM sodium azide and sealed with a coverslip before imaging on a Zeiss Axioplan 2 microscope. Samples were observed with a Zeiss Plan Neofluar 20x/0.50 Ph2 objective, images captured using a Zeiss AxioCam and the software AxioVision 4.8. AO or DAPI accumulation was imaged at 100msec exposure time, except for the strains CB7431 [bus-17(br2)] and AW1452 [bus-17(e2800)], where images were taken at 50msec exposure time.

Transfected cells having 1μg each of non-tagged receptors were rinsed twice in solution α (HBSS with 2.5mM probenecid, 250mM NaOH, adjusted to pH 7.4 by HCl) 24 hrs after transfection. Loading of cells with cell permeant Fluo-4, AM (Invitrogen) was done at 1 μM Fluo-4, AM diluted in solution α containing 0.003% Pluronic® F-127 (Invitrogen) for 30 mins at room temperature. The cells were rinsed twice in solution α and allowed to incubate at room temperature for 30 mins before fluorescence signal was monitored. Fluorescence signal was measured on an LSM 710 NLO Confocal Laser Scanning Microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan-Neofluar 20x/0.50 Ph2 objective. The machine was set to excite the samples through an argon laser (LASOS Lasertechnik GmbH, Jena, Germany) at 488 nm and record 493–622 nm emission in a time series manner. The pinhole was set at 44μm and pixel dwell at 1.58 μs. An 8-bit frame was captured every two seconds for 150 seconds. Cell viability was assayed by stimulating the cells with 60 mM KCl at the end of the experiment. The changes in relative fluorescence unit (RFU) were calculated by selecting at least five regions of interest (ROI) from each experiment.