IFA was performed according to previous studies (20 (link), 24 (link), 27 (link)). Sf21 cells (1 × 104 cells/well) were seeded into the wells of Millicell® EZ chamber slides (Millipore, PEZGS0816) and infected with individual recombinant baculoviruses at multiplicity of infection (MOI)=1 for 2 days. The infected cells were washed once with DPBS and then fixed with 4% paraformaldehyde (Electron Microscopy Science, 15710) for 15 min at room temperature. After blocking with ChonBlock Blocking/Sample Dilution Buffer (Chondrex, 9068), the cells were incubated with primary mouse anti-His antibody (1:200; Bio-Rad, MCA1396) for 2 h at room temperature. After three washes with DPBS plus 0.1% Tween 20 (DPBST), the cells were incubated with Alexa Fluor 555 Goat anti-Mouse IgG (H+L) secondary antibody (1:5000; Invitrogen, A21422) and counterstained with 49,69-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, D1306). Distribution of fluorescence signal was observed under Zeiss laser confocal microscopy (LSM780).
Millicell ez chamber slides
Manufactured by Merck Group
The Millicell EZ chamber slides are a type of cell culture equipment designed for in vitro cell-based experiments. They provide a controlled environment for culturing and observing cells. The slides feature a built-in chamber that allows for the introduction and exchange of culture media, enabling researchers to monitor cellular behavior and responses under various experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (4 mentions)
Mitosox red reagent, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Chonblock blocking sample dilution buffer, by Chondrex (1 mentions)
Alexa fluor 555 goat anti mouse igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
5 brdu, by Merck Group (1 mentions)
Bz x700 microscope, by Keyence (1 mentions)
Evos fl fluorescent microscope, by Thermo Fisher Scientific (1 mentions)
H1399, by Thermo Fisher Scientific (1 mentions)
Rotenone, by Merck Group (1 mentions)
5 protocols using millicell ez chamber slides
1
Visualizing His-Tagged Proteins in Sf21 Cells
2
BrdU and PH3 Immunofluorescence Assay
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Cells plated on Millicell EZ chamber slides (Millipore, cat. no. PEZGS0416) were cultured in media containing 10uM 5-BrdU (Sigma-Aldrich, #B5002) for 5 h at 37 °C. Cells were then washed with PBS and fixed with 3% paraformaldehyde in PBS for 10 min. To denature DNA, cells were incubated with 2 N HCl in PBST for 1 h, washed with PBST, permeabilized in 0.1% PBST for 1–3 h, and blocked with immunofluorescence blocking buffer for 2–3 h. To detect BrdU and PH3, cells were incubated with anti-PH3 antibody in blocking solution at 4 °C overnight, washed with PBST, incubated with Alexa Fluor® 488 anti-BrdU antibody and Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG (H + L) for 2 h, washed with PBST, stained with DAPI, and mounted in ProLong Gold antifade mountant (Life Technologies, #P36930). Images were acquired with the Keyence BZ-X700 microscope.
3
Immunofluorescent Cleaved Caspase-3 Assay
4
Mitochondrial Superoxide Detection Using MitoSOX
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MitoSOX Red reagent (Thermo Fisher Scientific, M36008) was used to detect mitochondrial superoxide levels in live cells. MG‐63 cells were cultured in Millicell EZ chamber slides (EMD Millipore). A 5 mM MitoSOX Red reagent stock solution was made by dilution into dimethyl sulfoxide (DMSO). A 5 μM MitoSOX Red reagent working solution was made by diluting the stock into a culture medium. Cells were loaded with MitoSOX Red reagent by incubating for 10 minutes at 37°C protected from light. Hoechst 33342 (1:2000) live‐cell dye was used as a counterstain to detect the nuclei of live cells (Thermo Fisher Scientific, H1399). Cells were washed three times with a warm medium. Intensity measurements were obtained using the Zen Blue software (Zeiss) analyzed using Prism 8 (GraphPad). Rotenone (Sigma, St. Louis, MO, USA; R8875) was applied as a positive control. For each replicate (n = 6 replicates/condition), average ratios were derived from four different fields of views of 5 to 10 individual cells. Data are presented as mean ± SEM error bars; ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05 (one‐way ANOVA with Tukey's multiple comparisons test compared with vehicle).
5
Quantifying Mitochondrial Superoxide in Cells
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MitoSOX™ Red reagent (ThermoFisher, M36008) was used to detect mitochondrial superoxide levels in live cells. MG-63 cells were cultured in Millicell® EZ chamber slides (EMD Millipore). A 5mM MitoSOX™ Red reagent stock solution was made by dilution into dimethyl sulfoxide (DMSO). A 5μM MitoSOX™ Red reagent working solution was made by diluting the stock into a culture medium. Cells were loaded with MitoSOX™ Red reagent by incubating for 10 minutes at 37˚C protected from light. Hoechst 33342 (1:2000) live-cell dye was used as a counterstain to detect the nuclei of live cells (ThermoFisher, H1399). Cells were washed three times with a warm medium. Intensity measurements were obtained using the Zen Blue software (Zeiss) analyzed using Prism 8 (GraphPad). Rotenone (Sigma, R8875) was applied as a positive control. For each replicate (n=6 replicates/condition), average ratios were derived from four different fields of views of 5-10 individual cells. Data are presented as mean±SEM error bars; * * * * p ≤ 0.0001, * * * p ≤ 0.001, * * p ≤ 0.01, and * p ≤ 0.05 (one-way ANOVA with Tukey's multiple comparisons test compared to vehicle).
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