GUVs containing DOPC (89.5 or 69.5 mol%), DOPS (10 or 30 mol%), and the lipid fluorophore Atto 647N DOPE (0.5 mol%) were prepared in 270 mOsm sucrose using the polyvinyl alcohol (PVA)-gel hydration-based method as in Weinberger et al. (49 (link)). Briefly, lipids were mixed in chloroform at a total concentration of 1 mM. The 40-μL solution of the lipid mixture was spread on a 5% wt/vol PVA film dried on a 25 × 25 mm coverslip (VWR) and then put under vacuum for at least 2 h to form a dry lipid film. The dried lipid film was hydrated with 500 μL of 270 mOsm sucrose solution for 2 h at room temperature to produce GUV dispersion, which was collected and stored in a 1.5-mL microcentrifuge tube.
25 mm coverslip
Manufactured by Avantor
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The 25 × 25 mm coverslip is a flat, thin glass or plastic sheet used in microscopy to cover and protect specimens on a microscope slide. It serves to keep the specimen in place and prevent contamination.
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5 protocols using 25 mm coverslip
1
Preparation of Fluorescent GUVs
2
Preparation of Fluorescent Giant Unilamellar Vesicles
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GUVs containing DOPC (69.5 mol%), DOPS (30 mol%), and the lipid fluorophore Atto 647 DOPE (0.5 mol%) were prepared in 270 mOsm sucrose using PVA-gel hydration-based method as in Weinberger et al. (32 (link)). Briefly, lipids were mixed in chloroform at a total concentration of 1 mM. The 40 μL solution of the lipid mixture was spread on a 5% w/v PVA film dried on a 25 × 25 mm coverslip (VWR) and then put under vacuum for at least 2 h to form a dry lipid film. The dried lipid film was hydrated with 500 μL of 270 mOsm sucrose solution for 2 h at room temperature to produce GUV dispersion which was collected and stored in a 1.5-mL microcentrifuge tube.
3
GUV Preparation Using PVA-Gel Hydration
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GUVs containing DOPC (69.5 mol%), DOPS (30 mol%), and the lipid fluorophore Atto 647 DOPE (0.5 mol%) were prepared in 270 mOsm sucrose using PVA-gel hydration-based method as in Weinberger et al. (32 (link)). Briefly, lipids were mixed in chloroform at a total concentration of 1 mM. The 40 μL solution of the lipid mixture was spread on a 5% w/v PVA film dried on a 25 × 25 mm coverslip (VWR, Radnor, PA) and then put under vacuum for at least 2 h to form a dry lipid film. The dried lipid film was hydrated with 500 μL of 270 mOsm sucrose solution for 2 h at room temperature to produce GUV dispersion which was collected and stored in a 1.5 ml microcentrifuge tube.
4
Visualizing Osteoclast Dynamics with Ads and F-actin
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In order to visualize sub-cellular localization of Ads and F-actin as they assemble and disassemble, as well as podosome dynamics in living osteoclasts, RAW264.7 cells were seeded into 6-well plates with 25-mm coverslips (VWR International, Radnor, PA, USA) in each well. The cells were transiently cotransfected with pEGFP-Ads and pRFP-Lifeact using the FuGENE HD Transfection reagent according to the manufacturer’s instructions (version 11.0; Roche Applied Science, Madison, WI, USA). The transfected cells were incubated with sRANKL for 4 days. Time-lapse video and images were obtained using Leica confocal fluorescence microscopy (magnification, ×40; Leica Microsystems, Buffalo Grove, IL, USA).
5
Isolation and Immunostaining of Peritoneal Macrophages
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Peritoneal macrophage was isolated by following as per the IAEC approval (NISER/SBS/IAEC/AH-229). 4-6 weeks-old mice were euthanized, and macrophage cells were isolated from the peritoneal cavity in chilled 1XPBS (Phosphate buffer saline), as per the protocol (Mahish et al. 2023) (link). Further, cells were seeded on 25 mm coverslips (VWR) and supplemented with complete RPMI media (Invitrogen) supplemented with 10% Fetal Bovine Serum (Gibco) antibacterial-antifungal agents Penicillin-Streptomycin (HiMedia) and Amphotericin-B (Sigma). Cells were grown at 37 °C with 5% CO 2 in an incubator. Three biological repeats of the experiments have been performed. For experiment purpose, TRPM8 modulators were added for total 4 h (3:30 h alone and last 30 min with or without β-MCD where applicable). After incubation, cells were fixed with 4% PFA. For immunostaining, cells were washed thrice with 1XPBS and left unpermeabilized to study the surface expression only. Cell were further stained with TRPM8 antibody (1:500 dilution) overnight. Next day, the cells were washed with 1XPBS and incubated with anti-rabbit antibody AF-488 (1:1000) and with CTxB-594 (1:500) for 2 h. At last, the nucleus was stained in 1:1000 concentration for 15 min. Cells were mounted on the coverslip with the mounting agent Fluoromount-G (SouthernBiotech).
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