B16/F10 cells (3 × 105) were plated at 37 °C on eight-chamber culture slides (BD Biosciences) coated with C1q (20 μg ml−1), FN (20 μg ml−1), FN+C1q (20 μg ml−1 each) or poly-lysine (PolyLys; 70 μg ml−1) and left to adhere for 30 min. The cells were fixed and permeabilized with the FIX & PERM Cell Permeabilization Kit (Società Italiana Chimici, Italy), stained with N-(7-nitrobenz-2-oxa-1,3-diazol-4-y)-conjugated phallacidin (NBD-phallacidin) (Molecular Probes, Invitrogen 1:50 dilution) and mouse monoclonal anti-paxillin (Merck-Millipore, clone 5H11, 1:100 dilution) followed by Cy3-conjugated F(ab′)2 goat anti-mouse IgG (Jackson ImmunoResearch Catalogue number 115-166-062, 1:300 dilution) as previously described39 (link)66 (link). Images were acquired with the Leica TCS SP2 confocal system (Leica Microsystems) using the Leica Confocal Software and a 633 fluorescence objective on a Leica DM IRE2 microscope (Leica Microsystems) or with a Nikon C1Si confocal system, using the Nikon EZ-C1 Confocal Software and a 633 fluorescence objective on a Nikon TE2000-U inverted microscope (Nikon, Melville, NY).
Eight chamber culture slides
Manufactured by BD
Sourced in Canada, Sweden
Eight-chamber culture slides are a laboratory equipment designed to facilitate the culturing and observation of cells or tissues. These slides feature eight individual chambers, allowing for the simultaneous cultivation and study of multiple samples within a single device.
Automatically generated - may contain errors
Lab products found in correlation
Dmire2 microscope, by Leica (1 mentions)
Tcs sp2 confocal system, by Leica (1 mentions)
Nbd phallacidin, by Thermo Fisher Scientific (1 mentions)
Te2000 u inverted microscope, by Nikon (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Duolink image tool, by Olink (1 mentions)
Lsm 510 laser scanning fluorescence confocal microscope, by Zeiss (1 mentions)
Dmem medium, by Euroclone (1 mentions)
6 protocols using eight chamber culture slides
1
Microscopic Analysis of Cell Adhesion
2
Apoptosis Quantification via TUNEL Assay
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Cells were seeded into eight-chamber culture slides (BD Biosciences, Mississauga, ON, Canada) where they were transfected with either control or lincRNA-p21 siRNAs, and infected with adeno-GFP or adeno-ING1b viruses for a total of 48 h. Subsequently, TUNEL assays were performed using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Etobicoke, ON, Canada) according to the manufacturer's recommendations. Post processing, the cells were visualized using a Zeiss Axiovert 200 microscope and images were captured using AxioVision software.
3
Quantifying Endogenous Protein Complexes
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MDA-MB-231-BM cells in eight-chamber culture slides (Falcon) were washed in ice-cold PBS, fixed for 10 min in ice-cold acetone and washed again in PBS; in situ PLA was then performed at the PLA proteomics facility, SciLife Lab, Uppsala, Sweden, using the antibodies described in Supplementary Table S2 . The endogenous complexes, visualized by fluorescent dots, between HAS2 and USP17, or HAS2 and USP4, were counted by Duolink ImageTool (Olink Bioscience). As negative controls, one of the primary antibodies was omitted or rabbit IgG isotype control was used instead of anti-USP17.
4
Immunofluorescence Microscopy of MSTO-211H Cells
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MSTO-211H cells were seeded into eight-chamber culture slides (BD Falcon). The next day, cells were rinsed with ice-cold PBS buffer and fixed with 4% paraformaldehyde for 10′ at room temperature and then permeabilized with 1% Triton X-100. Cells were incubated overnight with the indicated antibody. The day after, cells were washed with cold PBS three times for 3 min each and stained for 2 h with a secondary antibody Alexa 488-conjugated goat anti-mouse IgG or anti-rabbit IgG (Molecular Probes Cells) and counterstained with DAPI (40,6-diamidino-2-phenylindole dihydrochloride). Cells were examined under a Zeiss LSM 510 laser scanning fluorescence confocal microscope (Zeiss, Wetzlar, Germany).
5
Immunofluorescence Staining of MSTO-211H Cells
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Briefly, MSTO-211H cells were seeded into eight-chamber culture slides (BD Falcon). The next day, cells were rinsed with ice-cold PBS buffer and fixed with 4% paraformaldehyde for 10′ at room temperature and then permeabilized with 1% Triton X-100. The cells were incubated overnight with the indicated antibody. The day after, cells were washed with cold PBS three times for 3 min each and stained for 2 hrs with a secondary antibody Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes Cells) and counterstained with DAPI (40, 6-diamidino-2-phenylindole, dihydrochloride). Cells were examined under a Zeiss LSM 510 laser scanning fluorescence confocal microscope (Zeiss, Wetzlar, Germany).
6
Fluorescent Labeling of miRNA-Nanocarrier Complexes
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Human Embryonic Kidney 293 cells (HEK 293T) were cultured in DMEM medium (Euroclone S.p.A., Pero, Milan, Italy, cat no ECM0102L) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 μg/mL streptomycin at 37°C in 5% CO2. Cells were plated on eight-chamber culture slides (Falcon cat no 354108) at a density of 2×104 cells/mL per well. After 24 h of incubation, cells were treated with polymers alone or CNT/miRNA complexes. FAM-mir-503 (50 nM) was incubated with pure polyamines and polyamine-coated CNTs at different weight ratios (polymer/miRNA ratios of 2:1, 4:1, 8:1, 10:1 w/w and CNT/miRNA 5:1, 10:1, 20:1 w/w, respectively) for 15 min at room temperature to allow complexation. Cells were incubated for 4 h with the complex formed by FAM-miR-503 and CNTs, washed with phosphate-buffered saline (PBS) and cultured for 24 h. After 24 h, cells were washed three times with cold PBS and fixed by incubation with a methanol/acetone (2:1) solution for 10 min at −20°C. After three PBS washes, cells were stained with 150 μL of a solution of phalloidin-TRITC (10 μg/mL) for 1 h, washed again and the nuclei stained with 150 μL of intercalating dye Hoechst 33258 (4 μg/mL). Fixed cells were mounted with a glycerol/PBS solution (3:1) and kept covered to prevent dye photobleaching until fluorescence image acquisition.
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