The Methods section for Cerny et al was published incorrectly. The entire section is reprinted below in its correct form.
Ionomycin treatment, β‐hexosaminidase release and glass bead wounding. For determination of released β‐hexosaminidase after ionomycin treatment, HeLa cells were plated for 2–3 days on six‐well plates, placed in a CO2/37 °C incubator and grown to confluency (less than 5% empty space between cells) using 3 μl DMEM medium supplemented with 10% fetal calf serum. One day before the experiment, the media were replaced with fresh pre‐warmed media (3 ml). Cells were treated, or not, with vacuolin (1 μM vacuolin/0.2% DMSO or 0.2% DMSO final concentration) as follows: 1 ml of medium was removed from each well and mixed with 3 μl of 3 mM vacuolin/DMSO and 3 μl DMSO or 6 μl DMSO; the mixtures were kept for 20 min at 37 °C and then gently added back to the corresponding wells. After a 2‐h incubation in the CO2/37 °C incubator, the media were carefully replaced with 1 ml of pre‐warmed HMEM media including 1% bovine serum albumin (BSA) and containing different combinations of ionomycin and vacuolin. This was followed by immediate transfer of the plates back to the CO2/37 °C incubator for an additional 10 min. The HMEM solutions were prepared by addition of 2.5 μl 2 mM ionomycin/DMSO or 2.5 μl DMSO into pre‐warmed (37 °C) HMEM. These HMEM‐containing solutions were supplemented with 1 μl vacuolin (3 mM vacuolin/DMSO) or 1 μl DMSO as needed. At the end of the 10 min period, the media were rapidly removed and centrifuged for 10 min at 1,200 rpm and 4 °C to eliminate cell debris. Released β‐hexosaminidase activity (Veldman et al, 2001) was determined by mixing 50 μl of these supernatants with 50 μl of 2.6 mM 4‐methylumbellyferyl N‐acetyl‐β‐d‐glucosaminidase (Sigma Co., St Louis, MO, USA) dissolved in 330 mM citrate/phosphate buffer, pH 4.8 (pre‐warmed to 37 °C) and 50 μl of distilled water (also pre‐warmed to 37 °C). The reaction was allowed to run for 10 min or less, stopped by a transfer of the mixture to ice. Appearance of the fluorescent product was determined with a fluorimeter using an excitation of 360 nm and emission of 450 nm. Total cellular β‐hexosaminidase was determined in parallel samples after permeabilization of the cells with 1 ml HMEM supplemented with 1% BSA and 1% NP‐40.
For the immunofluorescence experiments, BSC‐1 cells were plated for 2–3 days on six‐well plates containing a 25 mm diameter glass cover slip in their base. The cells were then grown to 70% confluency using 3 ml DMEM medium supplemented with 10% fetal calf serum. The protocol for treatment with vacuolin and ionomycin was identical to that above. After ionomycin stimulation, the six‐well plates containing the glass cover slips were transferred to ice water, washed once with ice‐cold PBS and then processed for immunofluorescence staining.
For cell wounding (McNeil & Warder, 1987), cells were plated in 100 mm diameter dishes and wounded with rolling glass beads, as follows: 100 mg of 425–600 μm diameter, acid‐washed glass beads (Sigma Co.) were mixed with 0.1 ml media and evenly sprinkled from an Eppendorf tube held 2 cm above the cells. The dishes containing the cell monolayers were gently rocked five times at 25 °C to allow rolling of the beads on top of the cells. After wounding, 0.9 ml of pre‐warmed HMEM media was added for a 5 min incubation at 37 °C in the CO2/37 °C incubator. The media from these cells were rapidly removed and centrifuged for 10 min at 1,200 rpm and 4 °C to remove cell debris, and the activity of the released β‐hexosaminidase was determined as described above.
Immunofluorescence imaging. For surface staining, the cells were incubated for 30 min at 4 °C (cold room) with antibodies specific for Lamp‐1 or desmoyokin‐AHNAK dissolved in HMEM with 1% BSA. Cells were then washed three times with ice cold PBS followed by fixation for 10 min with 3.7% paraformaldehyde (pre‐warmed to 37 °C), quenching for 10 min at 25 °C with 50 mM ammonium chloride in PBS+ and washed seven times with PBS+. Final staining was done using fluorescently labelled secondary antibodies and supplemented with Hoechst dye for nuclear staining when required. For total staining, the cells were washed three times with ice cold PBS and fixed as described, followed by permeabilization for 5 min at 25 °C with 0.05% saponin dissolved in HMEM with 1% BSA. Subsequent staining was done as for surface staining using the appropriate combination of antibodies. Images were acquired with a fully motorized wide‐field epifluorescence microscope (Axiovert 200M, Carl Zeiss, Inc., Thornwood, NY, USA) under the control of SlideBook (Intelligent Imaging Innovations, Denver, CO, USA). The microscope was equipped with phase contrast optics, motorized filter turret and lens holder for 20×, 63× and 100× lenses (Carl Zeiss, Inc.). Single‐optical fluorescent sections were captured with the 100× lens using a spinning‐wheel confocal head (Perkin‐Elmer, Boston, MA, USA) and the samples illuminated with a Griott 53 ion series laser. For wide‐field epifluorescence imaging, the samples were illuminated with a 175‐W Xenon lamp source (Sutter Instruments Co., Novato, CA, USA) optically coupled to the microscope with a liquid guide. SlideBook (Intelligent Innovations Inc.) was used to control image acquisition and data processing.
Laser‐mediated cell wounding and analysis of resealing. Cell wounding was performed with the infrared laser of a two‐photon microscope (LSM 510, Zeiss Inc.); analysis of resealing was on the basis of kinetics and the extent of FM1‐43 dye entry through open disruptions at 37 °C (Bansal et al, 2003; McNeil et al, 2003).
Supplementary information available at http://www.nature.com/embor/journal/v5/n9/full/7400243.html.
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