Cellmask plasma membrane stain

Thapsigargin was purchased from Sigma-Aldrich (Switzerland); cyclopiazonic acid (CPA) from Calbiochem; Fura-2/AM, Pluronic F-127 and CellMask Plasma Membrane Stain from Life Technologies (Carlsbad, CA). YFP–STIM1 was a gift from Dr Anant B. Parekh (University of Oxford, UK). YFP–STIM1L and YFP–STIM1LΔABD were constructed as described previously (Darbellay et al., 2011 (link)). RFP–STIM1 and RFP–STIM1L were created by gene synthesis, replacing YFP with RFP in both constructs (GeneCust; Dudelange, Luxembourg). YFP–STIM1ΔK was obtained from Addgene (Plasmid 18861; Cambridge, MA) and YFP–STIM1LΔK was obtained by mutagenesis (GeneCust; Dudelange, Luxembourg), Orai1 was obtained from Addgene (Plasmid 12199; Cambridge, MA) and pCMV/myc/ER/GFP (KDEL–GFP) was purchased from Life Technologies (Plasmid V823-20). D1_ER was kindly provided by Drs Amy Palmer and Roger Tsien (University of California, San Diego, CA; Palmer et al., 2004 (link)). Orai1–RFP was provided by Drs Dalia Al-Ansary and Barbara Niemeyer (Saarland University, Homburg, Germany; Quintana et al., 2011 (link)).

To determine the subcellular location of V-ATPase, cells were double stained targeting both the pump and cell boundaries. CP-A, OE33, and SK-GT-4 cells were fixed in methanol, and OACM5.1C cells were fixed in 3% PFA. Cells were incubated with primary antibody (1:50 Goat polyclonal antibody against human V-ATPase subunit a, Santa Cruz) in 1% PBS-BSA followed by incubation with secondary antibody (1:500 Alexa fluor 488, Molecular Probes). Cells' boundaries were determined using antibodies against a pool of cytoqueratins (Dako) in CP-A and SK-GT-4 cells or E-Cadherin (Dako) in OE33 cells and subsequently incubated with secondary antibody (1:1000 Alexa Fluor 546, Molecular Probes). Alternatively, OE33 edges were labeled using Cell Mask plasma membrane stain (Life Technologies) and OACM5.1C limits were labeled with Phalloidin Alexa 546 (Molecular Probes).
Cells were mounted in Fluoromount G (Southern Biotech, Birmingham, USA) + Draq5 (Abcam, Cambridge, UK) and images were recorded with a confocal laser scanning microscope (Leica TCS SP2) with a 63x objective.

The An. stephensi mosquito strain SDA500 was maintained at 26°C and 50–70% relative humidity under 13-h light/11-h dark conditions. Female BALB/c strain mice were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan). The P. berghei ANKA 234 strain was maintained by cyclical passages through BALB/c mice and An. stephensi mosquitoes. The tissues of mosquitoes were observed under the inverted microscope, IX73 equipped with a DP73 digital camera (Olympus, Tokyo, Japan).
Regarding fluorescent staining, the dissected salivary glands were immediately stained with 5 μg/l CellMask Plasma Membrane Stain (Life Technologies, Carlsbad, CA, USA) in phosphate-buffered saline (PBS, pH 7.2) for 10 min, and then washed three times. The specimens were mounted in PBS with Hoechst 33342 (NucBlue Live Cell Stain ReadyProbes Reagent, Life Technologies) and observed using the confocal microscope Leica TCS SP5 (Leica, Wetzlar, Germany).

U87MG cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Welgene, Gyeongsan, South Korea, and PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA and PAN Biotech) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA, and PAN Biotech). Cells were grown at 37 °C in a humidified atmosphere of 5% CO₂. Mycoplasma testing was conducted periodically. Cells were subjected to transient transfection with expression plasmids using the Effectene Reagent (QIAGEN, Hilden, Germany) or the Neon transfection method (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers’ manual. The following reagents were used at doses indicated in the figure legends: Rapamycin were obtained from Invivogen, Hong Kong; recombinant rat PDGF–BB (520-BB-050) was purchased from R&D Systems, Minneapolis, MN, USA; chlorpromazine was purchased from Sigma-Aldrich, St. Louis, MO, USA; Pitstop 2 was purchased from Abcam, Cambridge, United Kingdom; and CellMask Plasma Membrane Stain was purchased from Life Technologies, Carlsbad, CA, USA.

Oocytes were washed with M16 media three times and stained with CellMask™ Plasma Membrane Stain (2.5 μg/ml; C10046, Life technologies), BODIPY 500/510 dodecanoic acid (10 μg/ml; D-3823, Invitrogen), or ER Tracker™ Red dye (1 μg/ml; E34250, Invitrogen) for 30 min. The oocytes were rinsed with fresh M16 media three times and transferred to a glass bottom confocal dish. Live images of oocytes were obtained directly with a confocal microscope (Zeiss LSM710).

HT-29/B6 cells were seeded to the bottom of inverted filter supports (Millicell 12 mm; 0.4 μm pore size; Millipore; Germany) and imaging was performed on day 7 after confluency. Cells were washed once with HEPES/Ringer buffer (containing 2 g/l glucose, 2 mM probenecid, 1% penicillin/streptomycin). Subsequently, cells were loaded with the calcium dye Fluo-4-AM (4 mM Fluo-4-acetoxymethyl ester, Invitrogen) and the nucleus stain Hoechst 33342 (2 μM, Thermo Fisher Scientific, Waltham, MA., USA) for 1 h at 37 °C. For visualisation of cell borders 5 μg/ml CellMask plasma membrane stain (Invitrogen) was added. After washing thoroughly (3 × 5 min) with buffer, a first image was taken by confocal laser-scanning microscopy using a 40x water immersion objective (Zeiss LSM780, Jena, Germany) and maintained for 26 min with HlyA. After this first incubation period, images were taken every 2 min throughout the experiment with HlyA. The incubation chamber was heated to 37° and maintained with 5% CO₂ in ambient air throughout the experiment.