OvCa cells were stimulated with 10 μg/mL LPA with and without CC (5 µM) for 18 h in 8-well LabTek chamber glass slides (ThermoFisher) as described previously [13 (link),14 (link)]. Cells were fixed with 4% (w/v) paraformaldehyde for 20 min, washed 3 times using phosphate-buffered saline (PBS), permeabilized with 0.5% (v/v) Triton X-100 in PBS, and blocked for 20 min at 37 °C with tris-buffered saline with 0.5% bovine serum albumin (BSA), 0.1% glycine, and 0.05% Tween 20 (TBS-BGT). Cells were incubated for 1 h at 37 °C with primary antibody against phosphorylated p65RelA diluted in TBS-BGT followed by washing 3 times with TBS-BGT and incubation with Alexafluor-594 or -488 donkey anti-rabbit secondary antibody for 1 h at room temperature. Slides were mounted in Fluor Gel II (Invitrogen) containing 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired from five fields/well with two replica per experimental condition at 40× magnification using Olympus IX-70 fluorescence microscope (Olympus, Tokyo, Japan). Digital images were analyzed using ImageJ software (64-bit bundled in Java 8 for Windows, NIH, Bethesda, MD, USA).
Labtek chamber glass slides
Manufactured by Thermo Fisher Scientific
Sourced in United States
LabTek chamber glass slides are a type of laboratory equipment used for microscopy and cell culture applications. These slides feature a pre-fabricated chamber that allows for the containment and observation of samples. The slides are made of high-quality glass and provide a transparent surface for optimal visibility during microscopic examination or cell culturing.
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Lab products found in correlation
Ix70 fluorescence microscope, by Olympus (1 mentions)
Axiocam mr3, by Zeiss (1 mentions)
Mowiol, by Merck Group (1 mentions)
Axiovert z1, by Zeiss (1 mentions)
Goat anti human igg fitc, by Jackson ImmunoResearch (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Zen 2009 imaging software, by Zeiss (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
6 protocols using labtek chamber glass slides
1
Immunofluorescence Analysis of NF-κB Activation
2
Adenoviral-Mediated Hypoxia-Reoxygenation Model
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NCM were infected with adenoviruses expressing LacZ or Perm1 at an MOI of 50. Forty-eight hours after infection, the media were changed to DMEM without glucose, glutamine, and pyruvate. NCM were cultured in normoxic (5% CO2-95% air) or hypoxic (5% CO2-95% N2) conditions for an additional 4 h, after which media was replaced with DMEM containing 5 mM glucose. Cells were then cultured in normoxic conditions for 2 h to subject them to reoxygenation. Finally, at the termination of the culture period, media was collected and analyzed for LDH, using a commercial assay kit (Roche, 04744926001).
Cells incubated in the above manner were also subjected to TUNEL staining (Thermo Fisher, A23210). Briefly, NCM were plated on Lab-Tek chamber glass slides (Thermo Fisher), treated in the above manner and washed with PBS, followed by fixation in 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS, the cells were incubated with a TUNEL reaction buffer for 1 h at 37 °C in a humidified chamber. The percentage of CM with DNA nick-end labeling was determined by counting cells exhibiting yellow-green nuclei among 500 nuclei in triplicate plates in two independent experiments.
Other experimental procedures are described in thesupporting information .
Cells incubated in the above manner were also subjected to TUNEL staining (Thermo Fisher, A23210). Briefly, NCM were plated on Lab-Tek chamber glass slides (Thermo Fisher), treated in the above manner and washed with PBS, followed by fixation in 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS, the cells were incubated with a TUNEL reaction buffer for 1 h at 37 °C in a humidified chamber. The percentage of CM with DNA nick-end labeling was determined by counting cells exhibiting yellow-green nuclei among 500 nuclei in triplicate plates in two independent experiments.
Other experimental procedures are described in the
3
Immunofluorescence Analysis of Extracellular Matrix in Fibroblasts
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Fibroblasts (7 × 103) were cultured in 8-well Lab-Tek™ chamber glass slides (Thermo scientific) (Waltham, Massachusetts, USA) with complete medium (DMEM) for 24 h. The medium was then replaced, and the cells were treated with 50 µM uvaol for 24 h. Cells maintained in DMEM under the same conditions were used as controls. After treatment, the cultures were washed with PBS, fixed with 100% methanol for 10 min, and subjected to an indirect immunofluorescence assay, as previously described [52 (link)]. In brief, cells were rehydrated in PBS and incubated for 1 h with PBS containing 1% BSA to block non-specific binding. Next, samples were incubated with primary specific anti-fibronectin, anti-laminin, or anti-collagen type I antibodies (1:50) for 1 h in a humidified chamber. They were then washed with PBS, and incubated with appropriate FITC-conjugated secondary antibody (1:200) for 45 min at room temperature. DAPI staining was used to visualize the nuclei. Immuno-stained samples were analyzed by fluorescence microscopy. The staining of each section was analyzed to obtain the mean optical density value (MOD), which represented the fluorescence intensity per pixel [53 (link)].
4
Immunofluorescence Imaging of CSPG4 in A375 Melanoma
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A375 melanoma cells (ATCC) were seeded in LabTek glass chamber slides (Nunc) at a density of 2 × 104 cells per well. Cells were washed in PBS and fixed in 4% formaldehyde PBS for 15 minutes and then incubated with PBS-T, 10% goat serum, 0.05% Tween for 25 minutes at room temperature. Anti-CSPG4 IgG1/IgE or control NIP antibodies were incubated for 45 minutes with A375 cells at a concentration of 10 μg/mL. Cell-bound antibodies were detected with a secondary goat anti-human IgG-FITC (Jackson ImmunoResearch) or goat anti-human IgE antibody conjugated to FITC (Vector Labs). Nuclei were stained in Hoechst dye for 3 minutes. All washing steps used PBS, except the final wash with dH20. Cells were then mounted in Mowiol (Sigma) mounting medium. Fluorescence microscopy was performed on a Zeiss Axiovert Z.1 (40× objective) upright microscope. AxioCamMR3 and AxioVision Software (Carl Zeiss) was used for acquisition and analysis.
5
Quantifying CD3 Expression in Vδ1 T Cells
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Expanded Vδ1 T cells were sorted into cells with high and low surface expression of CD3ε using a MoFlo XDP Cell Sorter (Beckman Coulter). The cell populations were subsequently incubated on poly l -lysine-coated 8-well Lab-Tek glass chamber slides (Nunc; Thermo Fisher Scientific) for 30 min at 37°C. The cells were fixed with an equal volume of 8% paraformaldehyde for 15 min at 37°C, permeabilized with 0.3% triton X-100 in PBS for 5 min at room temperature and then blocked with 3% bovine serum albumin in PBS for 30 min at room temperature. The samples were incubated with a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD3ε antibody (clone SK7, BioLegend, 1/50 dilution in 3% BSA/PBS) and incubated overnight at 4°C. After two washes in PBS, the slides were counter-stained with Hoechst 33258 (Molecular Probes) for 30 min at room temperature to visualize the nuclei. The slides were then imaged under 63× oil immersion with a Zeiss laser scanning confocal 510 microscope (Carl Zeiss, Hertfordshire, UK). The mean fluorescence intensity (MFI) of CD3 staining and Hoechst staining in individual cells was quantified using Zen 2009 imaging software (Carl Zeiss). The MFI of Hoechst served as an internal reference control between the different populations.
6
Quantifying Doxorubicin Uptake in A549 Cells
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The doxorubicin accumulation in the A549 cell assay was as described by van Wijngaarden et al. ( 2007), with slight modifications. Briefly, A549 cells were incubated for 24 h in medium containing doxorubicin (0.5, 1, or 5 μM), with or without meloxicam (50 μM), or celecoxib (50 μM). Cells (1.5 x 10 5 ) were seeded on 4-well Lab-Tek glass Chamber Slides (Nalge Nunc International Corp., Naperville, IL, USA) and incubated overnight, after which test materials were added and incubated for 24 h. Subsequently, the culture medium was removed and cells were washed thrice with PBS. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, washed thrice with PBS and mounted under glass coverslips with Vectashield mounting medium (Brunschwig, Amsterdam, Netherlands). The cells were examined for doxorubicin fluorescence using fluorescence microscopy with excitation at 465-495 nm and detection at an emission maximum of 515-555 nm. Intracellular doxorubicin was quantified using computerized image analysis. Cells were measured and values are reported as mean light intensity per cell ± SD, corrected for background.
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