Immortalized mouse podocytes were grown at 33 °C and cultured on 8 chamber slides (ibidi GmbH, Planegg/Martinsried, Germany). The cells were incubated with 5 μM Fluo-8 (Sigma-Aldrich, St. Louis, USA) in imaging buffer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4*7H2O, 25 mM HEPES, 6 mM Glucose) for 30 min at 37 °C. Afterwards they were kept in imaging buffer for 20 min at room temperature. Imaging was performed with a Confocal microscope LSM710/Axiobserver Z1(Carl Zeiss Microimaging, Jena, Germany) using an 40x objective. Pictures were taken every 0.78 secs. 2 μM CNO was added after 10 pictures for the immortalized mouse podocytes and 100 μM after 20 pictures for the primary glomerular cells. 3 μM Ionomycin was used as positive control and added to the immortalized cells after 280 pictures and after 130 pictures to the primary cells.
8 chamber slides
Manufactured by Ibidi
Sourced in Germany
The 8-chamber slides are a laboratory equipment designed for various cell culture applications. They provide a structured platform with eight individual chambers, allowing for the simultaneous observation and analysis of multiple cell samples or experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Fluo 8, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Papain, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
4 6 diamidino 2 phenylindole dapi, by BD (1 mentions)
Plan apochromat 63 1.4 oil, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 647 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
7 protocols using 8 chamber slides
1
Immortalized Mouse Podocyte Calcium Imaging
2
Cell Seeding and Enumeration
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Cells were trypsinized, suspended in medium containing 10% FCS and 30,000 cells were seeded to a polylysine-covered 8-chamber slides (Ibidi, Munich). After further incubation for 2 h, the cells were fixed with 4% paraformaldehyde/PBS and stained with 4′,6-Diamidin-2-phenylindol (DAPI). Thereafter, the cell number was determined using the Keyence software function Hybrid Cell Count.
3
Astrocyte Cultures for Insulin Receptor Studies
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Primary cortical astrocyte cultures were prepared from IR-flox, IGF1R-flox or IR/IGF1R double floxed newborn pups as previously described (19). Briefly, cortices were surgically dissected from newborn pups, and pooled for the following digestion in Hibernate-A media supplemented with 4 mg/ml papain (Sigma) and 33 U/ml DNase I (Sigma) on a shaker at 37 °C for 30 min. Dissociated cells were plated on a T75 flask and cultured in DMEM/F12 (Gibco) plus 10% FBS and 1× pen/strep (Gibco). Contaminating non-astrocytes were depleted by vigorously shaking the following day. Culture media was changed every 3 days. Upon confluency, astrocytes were trypsinized and replated onto 6-well plates (Corning) or 8-chamber slides (ibidi). To induce flox allele recombination, IR-flox, IGF1R-flox, and double floxed astrocytes were infected with adenovirus encoding Cre:GFP (1 × 109 GC/ml) overnight, and cultured for an additional 5 days before experiments. To generate the control cells, the same floxed astrocytes were infected with adenovirus encoding GFP alone. In studies assessing autophagy flux by mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) reporter, adenovirus encoding Cre or Luciferase were used to generate KO and control cells.
4
Immunofluorescence Imaging of Cell Proteins
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Cells grown on 8-chamber slides (ibidi, Munich, Germany) were washed in phosphate-buffered saline, fixed with 4% paraformaldehyde, permeabilised with Triton X-100, blocked in 5% bovine serum albumin for 1 h, and incubated with anti-NEK8 and β-catenin antibodies at 4 °C overnight. The cells were stained with secondary antibody conjugated with FITC or phycoerythrin for 1 h at room temperature (20–25 °C) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; BD Bioscience). The cells were imaged using a Zeiss 800 laser-scanning 800 confocal microscope (Carl Zeiss, Jena, Germany).
5
Histamine-Induced Live-Cell Imaging
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Confocal microscopy was performed using LSM 800 or LSM 780 microscopes (Carl Zeiss) equipped with a Plan-Apochromat 63 ×/1.4 oil immersion objective. Life-cell imaging was conducted at 37 °C in medium containing 20 mM HEPES on 8-chamber µ-slides (Ibidi). Cells were stimulated by addition of 500 µM histamine.
6
Visualizing ATP-induced Intracellular Dynamics
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Transiently transfected NSC-34 cells in 8-chamber µ-slides (Ibidi, Grafeling, Germany) (5 × 105 cells/0.2 mL/chamber) were washed twice with DMEM/F12 medium and pre-incubated in DMEM/F12 medium at 37 °C for 1 h. Cells with fluorescent aggregates were identified using a DM IBRE inverted microscope and TCS SP confocal imaging system at 37 °C. Cells were then incubated in the absence (basal) or presence of 5 mM ATP for 20 min at 37 °C with fluorescent and bright-field images captured every 30 s with Leica confocal software.
7
Visualizing Lipid Droplets and Proliferation in Cells
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Cells were seeded and differentiated on 8-chamber µ-Slides (Ibidi®, Gräfelfing, Germany). Differentiated cells treated with vehicle (DMSO) or 1 µM AZD3965 for 24 h were fixed in 4% PFA for 10 min at room temperature, permeabilized with 0.1% Triton™ X-100 in PBS, and blocked with 3% BSA in PBS for 1 h. Cells were, subsequently, incubated with Ki67 antibody (NBP2-22112; Novus Biologicals, Littleton, CO, USA) at a 1:500 dilution in PBS 3% BSA overnight at 4 °C. Cells were washed with PBS and incubated with goat anti-rabbit Alexa Fluor 647 secondary antibody (Invitrogen; Waltham, MA, USA) at 1:2000 dilution in PBS 3% BSA for 1 h. Lipid droplets were stained with BODIPY® (Invitrogen, Waltham, MA, USA) for 20 min at room temperature according to the manufacturer’s recommendations. Nuclei were stained for 20 min at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) in PBS 3% BSA, diluted at 1:5000 from a 1 mg/mL stock. Images were acquired on the Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) at a magnification of 630× with 3× zoom.
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