Human gingival fibroblasts (hGFs) were isolated from healthy gingival tissues obtained from the extracted third molars of 8 healthy donors similar to previously described methods [35 (link),36 (link),37 (link)]. Patients were informed prior to extraction about the procedure and protocols and gave their written consent. The study was performed in line with the Declaration of Helsinki and “Good Scientific Practice” guidelines of the Medical University of Vienna. The study protocol was approved by the Ethics Committee of the Medical University of Vienna (EK Nr: 1079/2019). After extraction, teeth were placed in sterile vials containing Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich®, St. Louis, MO, USA) nutrient medium supplemented with 10% fetal bovine serum (FCS; Sigma-Aldrich®, St. Louis, MO, USA), 100 U/mL penicillin and 50 µg/mL streptomycin (PS, Gibco®; Life Technologies, Carlsbad, CA, USA). To isolate hGFs, gingival tissue surrounding the tooth crown was cut out, minced, and distributed on Petri dishes in DMEM. Every 3 days, the medium was removed and replaced with a fresh portion of DMEM, and the growth of cells from the tissue pieces was observed under the light microscope. Cells in the passages between the 3rd and 6th were used in the experiments.
Fetal bovine serum fcs
Manufactured by Merck Group
Sourced in United Kingdom
Fetal bovine serum (FCS) is a cell culture media supplement derived from the blood of bovine fetuses. It provides a rich source of nutrients, growth factors, and other components essential for the growth and maintenance of cells in vitro.
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Lab products found in correlation
L glutamine, by Merck Group (2 mentions)
Pcdna3.1 gfp, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Maxiprep kit, by Thermo Fisher Scientific (1 mentions)
Dotap, by Lipoid (1 mentions)
Carboxymethyl β cyclodextrin, by Merck Group (1 mentions)
Opti mem 1 reduced serum medium, by Merck Group (1 mentions)
Smartpool, by Horizon Discovery (1 mentions)
Transit lt1 reagent, by Mirus Bio (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
On targetplus non targeting control pool, by Horizon Discovery (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
7 protocols using fetal bovine serum fcs
1
Isolation of Human Gingival Fibroblasts
2
Lipid-based Nanoparticle Transfection Protocol
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DOPE (1,2-dioleoyl-sn-glycero-3-phospho ethanol amine and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were purchased from Lipoid (Germany). Carboxymethyl-β-cyclodextrin, Pluronic-F12, cholesterol, DMEM, Fetal bovine serum (FCS), L- Glutamine and Opti-MEM I Reduced-Serum Medium were purchased from Sigma Aldrich (UK). pc DNA3.1-GFP and maxi prep kit were purchased from thermofisher scientific, UK, COS7 and SH-SY5Y cell lines were obtained from ATCC, UK. E coli, ampicillin containing agar plated, Luria-Bertaini and agarose were also provided by University of Sunderland. TransIT-LT1 liposomal transfection reagents were purchased from Mirus Bio LLC Madison, (USA), Promega QuantiFluor® ONE dsDNA System was purchased from Promega (UK).
3
Isolation and Culture of Primary Porcine Renal Epithelial Cells
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Animal experiments were approved by the supervisory authority (LAVES—Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) according to the recommendation of their Ethics Committee (LAVES, AZ 33.19-42502-04-16/2343) and conducted in compliance with the ARRIVE guidelines, the German animal welfare law, the German guidelines for animal welfare, and EU Directive 2010/63/EU. All experiments were performed in accordance with relevant guidelines and regulations.
Landrace pig kidneys were preserved on static cold storage since retrieval and during transport to the laboratory. Kidney biopsies were collected from the renal cortex and incubated for 1 h at 37 °C with collagenase type II (Gibco, Life Technologies Corporation, New York City, NY, USA). After biopsy digestion, the cell suspension was centrifuged, and the cell pellet was resuspended in CnT-Prime Epithelial Cell Culture Medium (CnT-Prime Medium) (CELLnTEC ADVANCED CELL SYSTEMS AG, Bern, Switzerland) supplemented with 2% fetal bovine serum (FCS) (Sigma-Aldrich, St. Loius, MO, USA), penicillin–streptomycin 2% (C.C.Pro, Oberdorla, Germany) and seeded onto 6-well plates. The medium was replaced every second day.
Landrace pig kidneys were preserved on static cold storage since retrieval and during transport to the laboratory. Kidney biopsies were collected from the renal cortex and incubated for 1 h at 37 °C with collagenase type II (Gibco, Life Technologies Corporation, New York City, NY, USA). After biopsy digestion, the cell suspension was centrifuged, and the cell pellet was resuspended in CnT-Prime Epithelial Cell Culture Medium (CnT-Prime Medium) (CELLnTEC ADVANCED CELL SYSTEMS AG, Bern, Switzerland) supplemented with 2% fetal bovine serum (FCS) (Sigma-Aldrich, St. Loius, MO, USA), penicillin–streptomycin 2% (C.C.Pro, Oberdorla, Germany) and seeded onto 6-well plates. The medium was replaced every second day.
4
ULK1 Inhibition in HEK293T Cells
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All cells were cultivated at 37 °C, 5% CO2 in a humidified area in Dulbecco´s Modified Eagle Medium (4,5 g/L D -glucose, 41965–039, Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FCS) (F0804, Sigma Aldrich), 100 U/ml Penicillin and 100 µg/ml Streptomycin (15140–122, Sigma Aldrich). HEK293T cells for immunofluorescence were seeded one day before the treatment with 1 × 105 per well. For knockdown of ULK1 for immunofluorescence analysis, HEK293T cells were transfected in 24-well plates using DharmaFECT1 (T-2001–02, GE Dharmacon) with 50 nM ULK1 siRNA (L-005049–00–0010, SMARTpool, ON-TARGETplus, GE Dharmacon) or 50 nM of the On-TARGET plus Non-targeting Control Pool (D-001810–10–20, GE Dharmacon) for 72 h. For inhibition of ULK1 in immunofluorescence analysis, cells were pre-incubated for 5 h with 30 µM ULK1 inhibitor MRT67307. For splicing assays, HEK293T cells were seeded in six-well plates with 2.5 × 105 cells per well. Before transfection, cells were pre-incubated for three hours with 30 µM of the ULK inhibitor MRT67307. Transient transfection was carried out by using the TransIT-LT1 reagent (Mirus Bio LLC) following the manufacturer’s instructions and described previously [23] (link). Cells were harvested for total RNA isolation 20 h after ULK inhibitor addition. MEF ULK1/DKO cells were reconstituted as described before [20] (link).
5
Establishing Murine Splenocyte Cultures
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C57BL/6 mice (female, 8-11 weeks old) were acquired from Envigo. RAG-/- mice and C57BL/6 mice on different congenic backgrounds (CD45.2+/+, CD45.1+/+, CD45.1+/-, CD90.1+/+, CD90.1+/-) were derived from in-house breeding under specific pathogen-free conditions at our mouse facility at the Technical University Munich, Institute for Medical Microbiology, Immunology and Hygiene. The performed animal experiments were approved by the district government of Upper Bavaria (Department 5—Environment, Health and Consumer Protection ROB- 55.2-2532.Vet_02-17-138).
Murine splenocytes were cultured in RMPI (Life Technologies, Cat #31870074) with 10% fetal bovine serum (FCS) (Sigma, Cat #F7524), 0.025% L-glutamine (Sigma, Cat #G8540), 0.1% HEPES (Roth, Cat #HN77.4), 0.001% gentamycin (LifeTechnologies, Cat #15750037), 0.002% penicillin/streptomycin (LifeTechnologies, Cat #10378016) and 15 ng/ml recombinant human (rh) IL-15 (Peprotech, Cat # 200-15). The Platinum-E packaging cell line (PlatE) was cultured in DMEM (Life Technologies, Cat #10938025) supplemented with 10% FCS, 0.025% L-glutamine, 0.1% HEPES, 0.001% gentamycin, 0.002% streptomycin. All cells were grown in a humidified incubator at 37°C and 5% CO2.
Murine splenocytes were cultured in RMPI (Life Technologies, Cat #31870074) with 10% fetal bovine serum (FCS) (Sigma, Cat #F7524), 0.025% L-glutamine (Sigma, Cat #G8540), 0.1% HEPES (Roth, Cat #HN77.4), 0.001% gentamycin (LifeTechnologies, Cat #15750037), 0.002% penicillin/streptomycin (LifeTechnologies, Cat #10378016) and 15 ng/ml recombinant human (rh) IL-15 (Peprotech, Cat # 200-15). The Platinum-E packaging cell line (PlatE) was cultured in DMEM (Life Technologies, Cat #10938025) supplemented with 10% FCS, 0.025% L-glutamine, 0.1% HEPES, 0.001% gentamycin, 0.002% streptomycin. All cells were grown in a humidified incubator at 37°C and 5% CO2.
6
Chicken Embryonic Fibroblast Cell Culture and Virus Propagation
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Chicken embryonic fibroblast cells (CEFs) were generated from ten-day-old mixed-sex specific pathogen free (SPF) Valo eggs (Valo Biomedia GmbH). CEFs were cultured in M199 medium (Life Technologies, Paisley, UK), supplemented with 5% fetal bovine serum (FCS) (Sigma, Dorset, UK), 100 units/mL of penicillin and streptomycin (Life Technology), 0.25 μg/mL Fungizone (Sigma, Dorset, UK), and 10% TPB (tryptose phosphate broth), (Sigma, Dorset, UK).Virus stocks were 3rd passage of the virulent MDV (GaHV-2: RB1B) and vaccine strain of MDV (CVI988/Rispens), propagated as cell-associated stocks in CEF for 72 hours. The virus stocks were titrated on fresh CEFs and the plaques were visualized using anti-gB mAb (HB-3) for staining. Commercial CVI988/Rispens vaccine virus (Nobilis Rismavac) was obtained from Intervet.
7
Flow Cytometric Analysis of Lymphocyte Populations
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Lymphocyte populations were analyzed by flow cytometry [16, (link)18, (link)19, (link)22] (link). In summary, cells were isolated from the spleen, thymus, and femur of 5-7-week-old mice and treated with red blood cell lysis buffer Hybri-Max TM (Sigma Aldrich, St. Louis, MO, USA; #R7757). The cells were resuspended in PBS (Thermo Scientific, Basingstoke, UK; #BR0014G) containing 5% Fetal bovine serum, FCS (Sigma Life Science, St. Louis, Missouri, United States; #F7524), and counted using a Countess™ II Automated Cell Counter (Invitrogen, Carlsbad, CA, United States; #A27977). Then, the cell suspension was diluted with PBS to get a final cell concentration of 2.5 x 10 7 cells/mL. Finally, surface markers were labeled with fluorochrome-conjugated antibodies and the cell populations were analyzed using flow cytometry.
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