NIH 3T3 cells were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % FBS and 2 mM Glutamax™. All cells were incubated in a 5 % CO2 humidified incubator at 37°C for 24 h before experiments. Cells were seeded onto Corning CellBIND 96 well plates (10,000 cells per well) or Nunc Lab-Tek II 8-chamber slides (20,000 cells per well) and were grown for 24 h at 37°C / 5 % CO2 before experiments.
Lab tek 2 8 chamber slide
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lab-Tek II 8-chamber slides are a laboratory equipment product designed for cell culture and microscopy applications. The slides feature 8 individual chambers that allow for the simultaneous culturing and analysis of multiple cell samples. The chambers are constructed with a durable polystyrene material and include a unique cell-adherent surface to support cell attachment and growth.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Dulbecco s pbs dpbs, by Thermo Fisher Scientific (1 mentions)
Giemsa, by Thermo Fisher Scientific (1 mentions)
Cellsens entry software version 1.5, by Olympus (1 mentions)
Fluormount g, by Southern Biotech (1 mentions)
Immu mount, by Zeiss (1 mentions)
Dii ldl, by Thermo Fisher Scientific (1 mentions)
5 protocols using lab tek 2 8 chamber slide
1
NIH 3T3 Cell Culture Protocol
2
Dose-Dependent Viability Assay for Astrocytes and BMEC
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Cells were plated onto 96-well plates at 5000 cells/well (astrocytes) or 2500 cells/well (BMEC) 100 μL per well, and allowed to incubate at 37 °C, 5% CO2 for 24 h. Media was then removed and cells treated with media spiked with metal or organic compounds in a serial dose dilution ranging from 0 μM to 1000 μM and incubated for an additional 24 h prior to viability assays. For images, cells were plated at 50,000 cells/chamber on Nunc Lab-Tek II 8-chamber slides, treated in the same manner as the plates. Medium was aspirated, the chambers washed with Dulbecco’s PBS (DPBS, Gibco) and cells fixed with ice-cold methanol for 5 min. After removing the methanol, the slides were air dried before being stained with Giemsa (Gibco) stain (1:20 dilution of stock) for 10 min followed by extensive washing with tap water. Images were taken with an Olympus BX61 Microscope with D72 camera and cellSens Entry Software (version 1.5) (Olympus America, Melville, NY, USA).
3
Immunocytofluorescent Analysis of HBEC2 Cells
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Immunocytofluorescent analysis was performed as previously described [52 (link)]. Twenty-four hours after culture in the presence or absence of 50 μM Apo9F on Lab-Tek-II 8-chamber slides (Nalge Nunc International, Rochester, NY, USA), HBEC2 cells were further cultured with or without 1.5% CSE for another 24 h. Cells were fixed with 2% (w/v) paraformaldehyde in PBS for 15 min at 37 °C. The cells were then incubated with 0.2% Triton X-100 with 0.2% Saponin in a blocking solution containing 3% IgG-free BSA (bovine serum albumin), 1% Gelatin and 2% normal donkey serum for 1 h at RT (room temperature) and further incubated with the following primary antibodies at 4 °C overnight: phosphorylation-specific antibody for ATM. The immunolabeled cells were detected using F(ab)2-fragments of respective secondary antibody conjugated to DylightTM-549 (Jackson ImmunoResearch, West Grove, PA, USA) and mounted with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G™ (SouthernBiotech, Birmingham, AL, USA) for nuclear staining.
4
Imaging HUVEC organelles and LDL trafficking
5
Filipin Staining of Cholesterol in Mouse Lung Endothelial Cells
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Mouse lung endothelial cells were seeded in a Nunc Lab-Tek II 8-Chamber Slide and treated with compounds for indicated time points. Cells were fixed with 4% paraformaldehyde for 1 h at room temperature and then permeabilized with 0.5% Triton X-100 (for protein immunostaining) for 10 min prior to blocking in a blocking buffer (3% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20) for 12 h. Cells were incubated with Filipin staining dye in the blocking buffer overnight at 4 °C. Cells were washed with PBS, mounted with Immu-mount, and were visualized by fluorescence microscopy at a 200× magnification (Leica microsystem, Germany). The 25-HC@DDAB was treated at a concentration of 0.1, 1, and 10 μM for 24 h.
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