Cells were plated onto 35 mm-glass-bottomed dishes (Greiner Bio-One) one day prior to the experiment and incubated on the microscope stage at 37°C in humidified 5% CO2. Two Carl Zeiss confocal microscopes were used (LSM710, AxioObserver and LSM780 AxioObserver) with Fluar 40x/1.30 NA Oil objectives. The 488 nm (ATOF set at 4%) line from an argon ion laser was used to excite the p65-EGFP fusion protein and emitted light between 490–540 nm was detected through pinholes set to 5μm. Image capture was performed using the Zeiss Zen 2010b software. Quantification of p65-EGFP nuclear fluorescence (or cytoplasmic fluorescence at t0) was performed using Cell Tracker (version 0.6) using region of interest analysis [62 (link)]. The data was exported as mean fluorescence intensity. TNFα-induced nuclear NF-κB responses in U2OS cells were less robust in comparison to previously described single cell oscillations in different cell types [11 (link), 46 (link), 53 (link)]. In particular, the first peak translocation had a relatively low amplitude (Fig 4 ). A cell was classified to have responded if >10% change in the nuclear NF-κB p65-EGFP level (in comparison to a basal unstimulated level at t = 0) was observed for >6 consecutive imaging time-points (equivalent to >12 mins).
35 mm glass bottomed dishes
Manufactured by Greiner
Sourced in Germany, United States
35 mm-glass-bottomed dishes are specialized laboratory equipment used for various cell culture and microscopy applications. These dishes feature a circular glass bottom that allows for high-quality imaging and observation of cells and cellular processes under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Zen 2010 b sp1, by Zeiss (3 mentions)
Lsm 780 axioobserver, by Zeiss (1 mentions)
Zen 2010b software, by Zeiss (1 mentions)
Axio observer, by Zeiss (1 mentions)
H3570, by Thermo Fisher Scientific (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Filipin, by Polysciences (1 mentions)
Sight ds qimc camera, by Nikon (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
5 protocols using 35 mm glass bottomed dishes
1
Visualizing NF-κB Dynamics in U2OS Cells
2
Live-cell Confocal Imaging of IκBα and p65
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Cells were plated onto 35 mm-glass-bottomed dishes (Greiner Bio-One) and incubated on the microscope stage at 37 °C in humidified 5% CO2. Several Zeiss confocal microscopes were used (LSM Pascal, Exciter, 510meta, 710 or 780) with fluar × 40 numerical aperture (NA) 1.3 or plan-apochromat × 63 NA 1.4 objectives and appropriate excitation and emission wavelengths for the two fluorophores. Image capture was performed using the Zeiss software, either ‘Aim version 4.2 utilizing the Autofocus macro68 (link)' on the 5-series microscopes or ‘Zen 2010b SP1' on the 7-series microscopes. Quantification of IκBα-eGFP fluorescent signal of whole cells was performed using region of interest analysis in ‘Zen 2010b SP1'. The data were exported as mean fluorescence intensity. For quantification of p65-mCherry fluorescence, Cell Tracker (version 0.6)69 (link)70 (link) was used to estimate mean nuclear and whole-cell fluorescence level, which was expressed as a nuclear to total ratio.
3
Imaging and Quantification of NF-κB Signaling
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Cells were seeded into 35 mm glass-bottomed dishes (Greiner) and imaged using several Zeiss confocal microscopes (LSM Pascal, Exciter, 710, 780, 880) with Fluar 40x NA 1.3 objectives. Nuclei were stained with Hoechst 33342 prior to imaging where indicated (Thermo Fisher H3570, incubation with 100 ng/mL for 20 min). Cells were maintained at 37°C in humidified 5% CO2 throughout image acquisition. Image capture used Zeiss software (Aim version 4.2, Zen 2010b SP1 or Zen 2.1 SP3 FP2). Quantification of IκB⍺-eGFP fluorescent signal of whole cells was performed using region of interest (ROI) analysis in Zen 2010b SP1 software. Normalised expression level was calculated relative to average cell fluorescence intensity prior to treatment. Quantification of dynamic parameters was performed by a custom script based on the ‘findpeaks’ function using Matlab 2020a. Exemplar image sequences were generated in Fiji (Schindelin et al., 2012 (link)). Quantification of p65 response amplitude used ROI measurement of fluorescent signal in the nucleus and cytoplasm of cells prior to stimulation and at the apex of the first peak. Nuclear and cytoplasmic fluorescence tracking of LMB treatment experiments used CellTracker software (Shen et al., 2006 (link)).
4
Filipin Staining of NPC1 Cells
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NPC1 or normal cells were seeded on 35-mm glass-bottomed dishes (Greiner Bio-one, Frickenhausen, Germany) at a density of 1 × 104 cells/dish and incubated overnight. After the medium was exchanged with fresh DMEM (900 μL), the treatment solutions (100 μL) were applied to the dish. After incubation for 24 h, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were stained with filipin (PolySciences, Warrington, PA, USA) (50 μg/mL) for 45 min at room temperature. The confocal laser scanning microscopic (CLSM) observations were performed on a FluoView FV10i (Olympus, Tokyo, Japan) equipped with a 60× water-immersion objective lens (N/A 1.2) and a diode laser.
5
Imaging Membrane Dynamics in Macrophages
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Cells were plated onto 35 mm-glass bottomed dishes (Greiner Bio-One, Monroe, NC, USA) and incubated on the microscope stage at 37 °C in humidified 5% CO2. For monitoring membrane permeabilisation, cells were incubated with PI (5 μg/ml). A Zeiss LSM710 (Oberkochen, Alemania) confocal microscope with a Plan-apochromat × 63 1.3 NA oil immersion and × 40 1.3 NA objectives was used to visualise release of IL-1β. Image capture was performed using the ‘Zen 2010b SP1' Zeiss software. Alternatively, macrophages were imaged with a Nikon Eclipse Ti microscope equipped with a 40 × /0.60S Plan Fluor objective and a digital Sight DS-QiMc camera (Nikon, Tokyo, Japan) and the NIS-Elements AR software (Nikon). Time-lapse microscopy images were quantified either with ImageJ (US National Institutes of Health, Bethesda, MD, USA) or Cell Tracker (version 0.6, Pittsburgh, PA, USA).42 (link) To quantify changes in plasma membrane dynamics over the time, macrophages were labelled with CTB-AF647 (1 : 1000 dilution) for 30 min at 37 °C and imaged using the Nikon Eclipse Ti microscope as stated above. Inverted fluorescence images converted to grey scale were used for quantification of the mean grey value as relative fluorescence units (RFUs) in different regions of interest of the plasma membrane of each cell using ImageJ (US National Institutes of Health).
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