Cell imaging was performed by plating HeLa cells or HEK293 cells in a concentration of 105 cells cm−2 on chambered coverslips (Nunc). When required, cells were transfected with 400 ng of expression plasmids using the Nanofectin transfection reagent (see above). Cells were rinsed with PBS 24 h after transfection, fixed and permeabilized with 0.1% Triton X-100. Nonspecific immunostaining was blocked by incubating the cells in 3% BSA in PBS with 0.1% TWEEN-20. The following antibodies were used: anti-Flag, mouse monoclonal IgG (M5, F4042, Sigma), secondary antibody, Alexa-Flour-488 coupled goat anti-mouse IgG (A11011, Life Technologies), anti-SHARP.2 [rabbit polyclonal, (18 (link))], secondary antibody, Alexa-Fluor-568 coupled goat anti-rabbit IgG, (A11011, Life Technologies), anti-MLL2/KMT2D, goat polyclonal (I18, sc68671, Santa Cruz), secondary antibody, Alexa-Fluor-488 coupled donkey anti-goat (A11055, Life Technologies). Pictures were taken using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu), and a 100-W mercury lamp (HBO 103W/2, Osram). The following filter set was used: Green, (Alexa-Flour-488) ex: HQ470/40, em: HQ525/50. For confocal microscopy a Leica TCS SP8-HCS microscope was used. Fluorophores were excited with the 488 nm and the 561 nm laser lines, respectively. Pictures were taken as ARY-sections in sequential scan mode.
Chambered coverslips
Manufactured by Thermo Fisher Scientific
Chambered coverslips are a type of laboratory equipment used to create a confined space for cell culture experiments. They consist of a transparent glass or plastic base with a raised chamber that can accommodate cells, media, and other experimental samples. Chambered coverslips provide a controlled environment for observing and studying cellular behavior under a microscope.
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Lab products found in correlation
C4742, by Hamamatsu Photonics (1 mentions)
Hbo 103w 2, by Osram (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
2 protocols using chambered coverslips
1
Fluorescence Imaging of Protein Localization
2
Culturing NIH 3T3 Cells for Microscopy
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NIH 3T3 cells (ATCC CRL-1658) were grown at 37 °C in a humidified atmosphere with 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; 11550356, Life Technologies), 1% Penicillin/Streptomycin (P4333, Sigma) and 1× Gibco GlutaMAX. For microscopy experiments, cells were seeded in LabTek chambered coverslips (155411, NUNC) at a density of 40,000 cells per well and transfected with Lipofectamine 2000 (11668019, Invitrogen) according to the manufacturer’s protocol.
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