Mouse immobilized podocyte cells were trypsinized and counted by hemocytometer, 5000 cells were seeded on 24-well plates precoated with collagen type IV (10 mg/ml). After 20 min incubation at 37 °C, nonadherent cells were removed by washing with 1× PBS three times, and the adherent cells were fixed in 4% formaldehyde and then stained with 5000× Hochest. After washing, the adherent cells were determined using imaging by microscopy using a Nikon T1-SAM equipped with a ×4 objective. After five independent pictures were captured, the cell number was counted by Image J software. All experiments were repeated in triplicate for three independent experiments.
T1 sam
Manufactured by Nikon
Sourced in Japan
The T1-SAM is a specialized lab equipment product from Nikon. It is designed to perform specific analytical functions within a controlled laboratory environment. The core function of the T1-SAM is to provide accurate and reliable data measurements, but further details on its intended use are not available.
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4 protocols using t1 sam
1
Quantifying Adherent Podocyte Cells
2
Transwell Cell Migration Assay
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Transwell cell migration were performed using cell culture inserts with 8.0-µm-pore size membranes (Corning, Cat#3422) following the manufacturer’s protocol. Cells (2 × 104) were seeded onto the top chamber with 2% charcoal-stripped FBS containing DMEM or PRIM 1640 media. In the bottom chamber, either 100 ng/ml EGF or vehicle in 2% charcoal-stripped FBS containing DMEM media was added as a chemoattractant. After 12 h incubation, cells migrating to undersurface of the chamber were fixed by 4% paraformaldehyde for 30 min and were stained by Hochest 33342 for 30 min. Migrated cells were counted under inverted microscope (Nikon T1-SAM).
3
Histopathological Analysis of Tissue Samples
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Spleens and femurs were removed and fixed with 4% paraformaldehyde for at least 24 hours. Next, the samples were embedded in paraffin and cut into thin sections (4 μm thick) for the next staining analysis. We applied the hematoxylin and eosin staining (H&E) to conduct the regular histopathological microstructure discrimination. To determine the specific molecular alteration, we performed the immunohistochemistry analysis (IHC) using the according antibodies: anti-Heme oxygenase-1 (HO-1), 1 : 1000; anti-Keltch-like ECH-associated protein 1 (KEAP 1), 1 : 1500; and anti-sirtuin 1 (SIRT 1), 1 : 500. The H&E slides were investigated under a microscope (Nikon, T1-SAM, Japan) adapted with a CCD camera (Nikon, DS-Ri2, Japan) while the IHC slides were scanned using an automatic digital slide scanner (Pannoramic MIDI, 3D HISTECH, Hungary). A quantitative analysis of the IHC pictures was performed using the IHC profiler plugin in the ImageJ software [16 (link), 17 (link)].
4
Oxaliplatin Resistance in Colorectal Cancer
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Human colon cancer cell line SW480 and human embryonic kidney cell line 293T were purchased from ATCC. To induce OXA resistant SW480 cells, the cells were seeded onto 24 well plate before chemotherapeutics treatment. Next, SW480 cells were cultured in the presence of 0.5 mM oxaliplatin (OXA) for 24 hours followed by three days in fresh medium without a drug. This procedure was continued for six months with drug concentration increase 0.4μM per month, and the nal concentration is 2.5 mM.
Both of SW480 and SW480/OXA cells were cultured in Dulbecco Modi ed Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution in a cell incubator at 37 °C, 5% CO 2 , with saturated humidity.
Hoechst 33342 staining Cells were seeded onto bronectin coated 12 well plate, after treatment, 5 mg/ml Hoechst 33342 staining solution (Solarbio, Beijing, China) was added to cells and incubated for 20 mins at room temperature. Then cells were washed with PBS for three time. Images were collected by Nikon T1-SAM.
Both of SW480 and SW480/OXA cells were cultured in Dulbecco Modi ed Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution in a cell incubator at 37 °C, 5% CO 2 , with saturated humidity.
Hoechst 33342 staining Cells were seeded onto bronectin coated 12 well plate, after treatment, 5 mg/ml Hoechst 33342 staining solution (Solarbio, Beijing, China) was added to cells and incubated for 20 mins at room temperature. Then cells were washed with PBS for three time. Images were collected by Nikon T1-SAM.
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