TUNEL analysis was performed according to the manufacturer’s instructions (Roche, #11684795910). In brief, the cultured cells were cultivated on CultureSlides (BD Biosciences) and treated with inhibitors for a set time. The cells were subsequently washed 3 times with ice-cold PBS and fixed in a freshly prepared fixation solution (4% paraformaldehyde). After 10 min of fixation, the cells were washed twice with PBS and permeabilized in a freshly prepared permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 10 min. Afterward, the cells were incubated with a TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. After washing twice with PBS, the samples were mounted using a ProLong antifade reagent (Molecular Probes, #36934) and were imaged using a Leica DMI 4000B microscope.
Culture slides
Manufactured by BD
Sourced in United States
Culture slides are a laboratory equipment used for the cultivation and observation of microbial or cell cultures. They provide a surface for growing and observing cells or microorganisms in a controlled environment.
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Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Dmi4000b microscope, by Leica (3 mentions)
Plan achromat 40 objective lens, by Zeiss (2 mentions)
Mitospy orange, by BD (2 mentions)
Fv1000 confocal microscope, by Olympus (2 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
G2 spirit biotwinmicroscope, by Thermo Fisher Scientific (1 mentions)
Lamp2, by Abcam (1 mentions)
Tcs spii, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lc3 primary antibody, by Cell Signaling Technology (1 mentions)
Gelatin, by Merck Group (1 mentions)
Laminin, by BD (1 mentions)
Type 1 collagen, by BD (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Fibronectin, by BD (1 mentions)
24 protocols using culture slides
1
Apoptosis Detection by TUNEL Assay
2
Imaging CD4+ T Cells with Confocal Microscopy
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Fixed cells were viewed and imaged with a Zeiss LSM780 confocal microscope and Plan-ACHROMAT ×40 objective lens (digital zoom used in closer images). CD4+ T cells were allowed to attach by gravity for 20 min onto culture slides (BD Bioscience) coated with poly-D-lysine, during which time they were also stained with MitoSpy Orange as described above. Attached cells were then washed twice, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were stained with DAPI (4,6-diamidino-2-phenylindol) for nuclear staining. Stacked images were then collected at intervals of 0.5 or 1 μm. All images were processed and analysed with ZEN 3.1 blue edition software.
3
Cellular Localization of Contrast Agents
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Fluorescence microscopy was used to identify the cellular localisation of the contrast agents. Cells were grown and labelled in culture slides (BD Biosciences) and the same immunostaining procedures as described for flow cytometry were applied. For imaging the lysosomes, a rabbit antibody against the Lysosomal-Associated Membrane Protein 2 (LAMP2, Abcam, Cambridge, England) was used in conjunction with secondary goat anti-rabbit antibody conjugated to Alexa-Fluor-488 (Life Technologies, Paisley, Scotland). Particles and lysosomes were imaged using confocal microscopy (Leica TCS SP II) using a pinhole size of 1 airy unit. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies) and imaged using wide-field fluorescence.
For transmission electron microscopy (TEM) cells were grown and labelled in 3.5 cm dishes, followed by fixation with 4% formaldehyde/2.5% glutaraldehyde, post fixation with 1% osmium tetroxide, dehydration and embedding in epoxy resin. Thin sections (70 nm) were then collected over copper grids containing a formvar support film. For analysis of the contrast agents, those were simply dispersed of over the grids containing the support film and allowed to dry. Samples were analysed with a FEI Technai G2 Spirit BioTwin microscope operated at 100 kV.
For transmission electron microscopy (TEM) cells were grown and labelled in 3.5 cm dishes, followed by fixation with 4% formaldehyde/2.5% glutaraldehyde, post fixation with 1% osmium tetroxide, dehydration and embedding in epoxy resin. Thin sections (70 nm) were then collected over copper grids containing a formvar support film. For analysis of the contrast agents, those were simply dispersed of over the grids containing the support film and allowed to dry. Samples were analysed with a FEI Technai G2 Spirit BioTwin microscope operated at 100 kV.
4
Imaging CD4+ T Cells with Confocal Microscopy
5
Autophagy Detection in Cell Cultures
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Cells were grown on CultureSlides (BD Biosciences) and exposed (or not) to 20 μM of 6r or ZFL for 24 h. To detect autophagic vacuoles, the cells were subsequently incubated with 0.05 mM of monodansylcadaverine (MDC) in PBS at 37°C for 10 min. Afterward, the cells were washed with ice-cold PBS, and the fluorescence intensities were obtained immediately using a Leica DMI 4000B microscope. For the immunodetection of LC3 puncta, the specimens were fixed with 4% paraformaldehyde, washed with ice-cold PBS, permeabilized with Triton X-100, blocked with 1% BSA, and subsequently incubated with the LC3 primary antibody (Cell signaling, #3868) for an additional 16 h. Bound antibodies were visualized by incubating the specimens with anti-rabbit secondary antibodies (Molecular Probe, #21207) for 2 h. To visualize the nuclei, DAPI was employed as a counterstain.
6
Culturing Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) (Lonza, Switzerland) were maintained in endothelial cell–based medium 2 (Lonza, Switzerland) supplemented with 10% fetal calf serum and growth factors (human epidermal growth factor, hydrocortisone, human recombinant fibroblast growth factor-beta, vascular endothelial growth factor, Insulin-like growth factor, Ascorbic acid, Heparin, FBS, and Gentamicin/Amphotericin-B) at 37°C at 5% CO2. Cells were grown in culture flasks or culture slides (BD, USA) pre-coated with 0.1% gelatin (Sigma, UK). Pre-coating was carried out by incubating flasks with 100 uL/cm2 of 0.1% gelatin at 37°C for 2 hours. PAF and PAF receptor antagonist (1-(N,N-Dimethylcarbamoyl)-4-ethynyl-3-(3-fluoro-4-((1H-2-methylimidazo[4,5-c]pyridin-1-yl)methyl)benzoyl)-indole, HCl (Calbiochem, Germany), (both from Millipore, Germany) were used for treatments. Both PAF and PAFR blocker were diluted in dH2O according to the manufacture instructions and aliquoted. PAF was stored in - 20°Ca and PAFR antagonist was stored in 4°C until further use.
7
MDCK and HeLa Cell Culture Conditions
8
Immunofluorescence Staining of Pancreatic Cancer Cells
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For immunofluorescence experiments, pancreatic cancer cells were cultured overnight on 2-well chamber CultureSlides (BD Biosciences, Bedford, MA, USA). Before staining, the cells were washed with cold PBS and fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C. After washing once with PBS, fixed cells were permeabilized in PBS containing 0.5% Triton X-100 for 10 min and then blocked with 10% goat serum (Invitrogen) in PBS at 4 °C. The blocked cells were then incubated with the primary antiserum in PBS containing 10% goat serum for 1 h at room temperature. After further washes with 0.1% Tween 20 in PBS, an Alexa Fluor 555 goat antimouse IgG secondary antibody or Alexa Fluor 555 goat antirabbit IgG (Invitrogen) secondary antibodies were applied for 1 h. After the final wash, slides were incubated for 1 h with DAPI (1 µg/mL in PBS) for nuclei staining and with FITC-labeled antipan cytokeratin (C11) (Sigma-Aldrich) for total cellular cytokeratin stain, washed with PBS, and finally mounted with the ProLong Antifade Reagent (Invitrogen). Slides were observed the next day under a Nikon TE2000U microscope equipped with epifluorescence optics (Nikon Europe, Amsterdam, Netherlands). Images were captured with a cooled CCD camera (DS-5Mc, Nikon).
9
Immunofluorescent Staining of Cultured Cells
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Immunofluorescent staining was performed as previously described32 (link). In brief, the cells grown on CultureSlides (BD Biosciences) were exposed to the indicated reagents, fixed with 4% paraformaldehyde, washed twice with ice-cold PBS, permeabilised with 0.1% Triton X-100, and then blocked with 1% BSA for 1 h at room temperature. Subsequently, the cells were incubated with the indicated primary antibody for 16 h at 4 °C. After washing with PBS to remove unbound primary antibodies, bound antibodies were visualised by incubating the cells with secondary antibodies (Molecular probes). 4′,6-Diamidino-2-phenylindole (DAPI) was used as a counterstain to visualise the nuclei. The fluorescent images were obtained using a Leica DMI 4000B microscope with appropriate filters and lasers. For confocal analysis, the images were obtained using a FV1000 confocal microscope (Olympus) with a 60 × oil immersion lens, NA 1.35 (Uplsapo).
10
GCRV-JX01 Virus Internalization Assay
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Cells were plated onto CultureSlides (BD, Falcon) for 12 h. Then cells were pretreated with 20 mM ammonium chloride (NH4Cl) in water, 50 μM dynasore in DMSO or DMSO for 1 h at 4 °C. Cells were then adsorbed with GCRV-JX01 virions at an MOI of 20. After 1 h of viral adsorption at 0 °C, warm medium with inhibitors described above was quickly added. All inhibitors were present throughout all the experiment. At 30 min post-infection (mpi), non-internalized viruses were removed and washed three times with PBS. Then cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 10 min at room temperature. Viral particles were detected with mouse antiserum raised against GCRV-JX01-VP5 and counterstained with FITC-conjugated anti-mouse antibodies (Sigma). Coverslips were mounted using Vectashield hard Set with DAPI (Thermo Fisher Scientific). The staining patterns of the cells were examined by confocal microscopy as previously described [24 (link)].
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