Autophagy flux was analyzed with the pBABE-puro mCherry-EGFP-LC3B reporter plasmid purchased from Qiagen and previously described [58 (link)]. PC-3 cells were seeded in a 6 well plate. 24 hours after seeding cells were transfected with 1μg of plasmid for 20-24 h with 3 μL GeneJuiceTM (Novagen) following the manufacturer's protocol. The cells were washed and directly transfected with the indicated siRNA as described above. After 16 h the cells were washed again and seeded at subconfluence in μ-Slides 8-well (Ibidi). Images were acquired by fluorescence microscopy and the analyses were performed using ImageJ software. Micrographs of the cells were acquired in several focal planes and the analysis performed on the stacked images. Puncta structures mCherry-positive and expressing or not EGFP were quantified in more than 50 cells per condition. The proportion of autophagosomes was expressed as the percent of puncta with both colors.
μ slides 8 well
Manufactured by Ibidi
Sourced in Germany
The μ-Slides 8 Well is a microscope slide with eight individual wells designed for cell culture and analysis. It provides a standardized format for performing multiple experiments or observations simultaneously. The slide is made of high-quality materials and is suitable for a variety of microscopy techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Dmi6000b, by Leica (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Las af software, by Leica (1 mentions)
Dmi6000csb sp8, by Leica (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Liveblazer fret b g loading kit, by Thermo Fisher Scientific (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
4 protocols using μ slides 8 well
1
Quantifying Autophagic Flux in PC-3 Cells
2
Quantifying Neuronal Differentiation and Neurite Network Density
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Cells were plated on μ‐Slides 8 Well (ibidi GmbH, Munich, Germany) and treated as described in the sections above. Ten days after the initiation of differentiation, cells were fixed in 4 % formaldehyde (Sigma‐Aldrich) for 15 min; 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI, Sigma‐Aldrich) was added for 5 min at 1 μg/ml in PBS before three times washing with PBS. Microscopy was performed on an inverted microscope (DMI6000B, Leica, Wetzlar, Germany). Viable cells were counted in randomly selected areas of 1,300 × 1,000 μm in four replicates each. Counting was done blinded to the treatment condition and repeated three times. For neurite tracing, the “Simple Neurite Tracer” plugin for ImageJ by Mark Longair was used (http://fiji.sc/Simple_Neurite_Tracer ). For neurite network density measurement, optical density was measured using ImageJ and converted to a scale of 0 (no neurites) to 100 (total surface area covered with neurites). Sample names were blinded to the experimenter by random numbers before statistical analysis.
3
Confocal Microscopy of Cancer Cell Lines
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For confocal laser scanning microscopy, the epithelial human cancer cell line A431 (DSMZ No. ACC 91) and the human lung carcinoma epithelial cell line A549 (DSMZ No. ACC 107) were routinely propagated in Dulbecco's modified Eagle's medium (DMEM), with 2 % glutamine, penicillin/streptomycin (all from Gibco BRL, Eggenstein, Germany), and 10 % fetal calf serum (FCS, Biochrom AG, Berlin, Germany) at 37 °C with 5 % CO2 and were subcultured twice a week. For confocal microscopy, 27 000 cells were seeded in each well of a μ‐Slides 8 Well (ibidi GmbH, Martinsried, Germany) and were cultured at 37 °C for 24 h. Thereafter, dye‐labeled test substances were added to the cells at a final concentration of 1 μm. Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63×/1.4 HC PL APO CS2 oil immersion objective at 37 °C using the manufacture given LAS X software. Images of different groups were acquired with the same laser and detector settings by using the Leica LAS AF software. The fluorescence detection was performed sequentially for each channel set with the acousto optical beam splitter between λ=570 and 648 nm for the ICC dye and between λ=650 and 749 nm for IDCC dyes. ICC was excited using the λ=561 nm diode‐pumped solid‐state laser line, whereas IDCC dyes were excited with a λ=633 nm HeNe laser.
4
YopE Translocation Assay in Yersinia
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The YopE translocation assay was performed to examine the YopE secretion ability of Y. pseudotuberculosis strains carrying complementation constructs (pGM930) and pMK-bla (YopE-TEM, [89 (link)] using LiveBLAzer-FRET B/G Loading Kit (Life Technologies, Carlsbad, USA). Strains were pregrown in LB containing 0.1% (w/v) L-arabinose, 1 mM CaCl2, ampicillin and kanamycin for 2 h at 25°C. The bacteria were then shifted to 37°C, incubated for additional 2 h, washed and adjusted in PBS buffer to an OD600 of 1. HEp-2 cells (1.7x104) seeded in μ-Slides (8-well, Ibidi, Gräfelfing, Germany) were infected with Y. pseudotuberculosis strains at MOI of 50 and centrifuged for 5 min (400 g, RT). This was followed by incubation for 60 min at 37°C and three washing steps of the cells with PBS buffer. Then, 200 μL of infection buffer (RPMI, 20 mM HEPES, 0.4% BSA) containing gentamicin (25 μg/mL) were added and HEp-2 cells were stained with CCF4-AM loading dye according to the manufacturer’s protocol. YopE-TEM translocation was visualized using a fluorescence microscope (BZ-9000 Fluorescence Microscope, Keyence, Osaka, Japan).
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