Culture conditions of conditionally immortalized human podocytes have been described previously28 (link). Under permissive conditions at 33 °C, podocytes proliferated. Cultivation at 37 °C led to inactivation of the SV40 T-antigen to induce cell differentiation. Culture medium for human podocytes was RPMI 1640 Medium (Roth, Karlsruhe, Germany) with 10% fetal calf serum (FCS; PAA Laboratories, Pasching, Australia), 1% Penicillin/Streptomycin and 0.1% human insulin (Sigma-Aldrich, St. Louis, MO). Human glomerular mesangial cells (Cell systems, Kirkland, WA) proliferated at 37 °C in RPMI 1640 Medium (Roth, Karlsruhe, Germany) with 10% fetal calf serum (FCS; PAA Laboratories, Pasching, Australia), 1% Penicillin/Streptomycin and 0.1% human insulin (Sigma-Aldrich, St. Louis, MO). Human glomerular microvasculature endothelial cells (Cell systems, Kirkland, WA) were cultivated on CSC complete media (Cell systems, Kirkland, WA). To the medium, Bac-Off® (antibiotic) and CultureBoost-R™ (human recombinant growth factors; Cell systems, Kirkland, WA) were added, culture conditions were 37 °C and 5% CO2.
Rpmi 1640 medium
Manufactured by Carl Roth
Sourced in Germany
RPMI 1640 Medium is a cell culture medium commonly used to support the growth and maintenance of a variety of cells, including human and animal cell lines. It is a well-balanced formulation that provides the necessary nutrients and growth factors for cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
Human insulin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 protocols using rpmi 1640 medium
1
Culturing Immortalized Human Kidney Cells
2
Murine Lymphoma Cell Line for Telomere Analysis
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Murine DBA/2 lymphoma cell line L178Y-R (European Collection of Authenticated Cell Cultures via Merck KGaA, Darmstadt, Germany; LOT06/F/004) with known telomere length (79.7 kb) has been used in the present research as a reference for the Q-FISH analysis of the rainbow trout cells and to assess length of the trout telomeric DNA The culture was carried using RPMI1640 medium (Carl Roth GmbH) supplemented with 10% fetal bovine serum (Merck KGaA) and penicillin–streptomycin solution (50u and 0.05 mg per 1 ml of medium respectively; Merck KGaA) and maintained between 3 × 104 to 7 × 105 cells per ml. Incubation was carried out at 37 °C with 5% CO2 and continuous shaking at 90 rpm (New Brunswick™ S41i Incubator, Eppendorf AG) in 125-ml single use, vented, PETG Erlenmeyer flasks (Thermo Fisher Scientific Inc.). About 72 h prior to preparation of the interphase spreads, the cells were seeded to 50 ml of fresh culture medium at 106 cells per 1 ml. Incubation was conducted as mentioned above. After 72 h, the whole volume of the culture was centrifuged at 1000 rpm. Interphase spreads were prepared in the same way as the head kidney cells, despite the hypotonization step which lasted 45 min.
3
Podocyte Differentiation and Damage
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Human ciPodocytes (kindly gifted from Moin Saleem; Children’s and Renal Unit and Bristol Renal, University of Bristol) were proliferated under permissive conditions at 33 °C. Undifferentiated podocytes were dissociated using 1× trypsin-EDTA for 5 min at 33 °C, and 3.5 × 103 cells per cm2 were seeded onto uncoated, or chromium-, platinum- and laminin 511 (RLS 115 biogenes; 2.5 µg/mL)-coated glass slides, or plastic cover slides. When cultivated at 37 °C, the SV40 large T-antigen was inactivated for terminal cell differentiation. Podocytes were differentiated on glass cover slides in RPMI 1640 Medium (Roth, Karlsruhe, Germany), supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 0.1% insulin. After 7 days at 37 °C, cells were starved with 1% fetal calf serum overnight, and either left untreated for another 48 h, or treated with 5 ng/mL TGF-β for 48 h, to induce cell damage before fixation and imaging.
4
Podocyte Cytoskeletal Reorganization Assay
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Immortalized cultured human podocytes were proliferated under permissive conditions at 33 °C. When cultivated at 37 °C, the SV40 T-antigen was inactivated for cell differentiation. Podocytes were differentiated for 10 days on cover slides in RPMI 1640 Medium (Roth, Karlsruhe, Germany) with 10% fetal calf serum, 1% Penicillin/Streptomycin and 0.1% Insulin. At day 7 fetal calf serum was replaced to 10% control serum or patient serum. Cells were fixed at 0 h and 6 h using ice-cold methanol at − 20 °C for 10 min and permeabelized using 0.1% Triton for 10 min. After blocking with 10% donkey serum, immunofluorescent labeling of F-actin was done by incubation with Alexa Fluor 546 phalloidin (Invitrogen) at 4 °C overnight. Nuclei staining was done with Hoechst. Sides were mounted on glass slides and were visualized under fluorescent microscopy. For quantification of changes in actin cytoskeleton of podocytes, we used a scoring system published earlier14 (link)–16 (link): Type A: more than 90% of cell area filled with thick cables; type B: at least 2 thick cables running under nucleus and rest of cell area filled with fine cables; type C: no thick cables, but some cables present; type D: no cables visible in the central area of the cell.
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