Western blotting was performed as previously described [33 (link)]. In brief, human GCs were cultured on 60 mm dishes (Sarstedt, Nürnbrecht, Germany) in DMEM/Ham's F12 media supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin. To detect possible small differences in loading and to correct the results accordingly, β-actin served as internal standard. Some antibodies were also used for immunohistochemistry. For control purposes, some antibodies, including proNGF, were pre-incubated with a blocking peptide. This antibody was also used for immunohistochemistry. Western blot bands were visualized with chemiluminescence reagents (Pierce – Thermo Scientific, Waltham, USA). For the list of antibodies and blocking peptides used, please see Tables 1 and 2 . Human GCs were incubated with recombinant proNGF (Cedarlane, Burlington, Canada) and recombinant NGF (Alomone Labs, Jerusalem, Israel) each with 50 ng/ml for 1 h or 24 h in DMEM/Ham's F12 media without FCS, to examine downstream effects on early growth response 1 (EGR1) and choline acetyl-transferase (CHAT) level exerted by the factors. Bands were analyzed with ImageJ Software (National Institute of Health, Bethesda, MD, USA; version 1.45s) and the results normalized to the untreated control.
60 mm dishes
Manufactured by Sarstedt
Sourced in Germany
The Sarstedt 60 mm dishes are circular cell culture vessels made of high-quality polystyrene material. They provide a standardized surface area for cell growth and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Glutamine, by PAN Biotech (1 mentions)
Penicillin, by PAN Biotech (1 mentions)
Streptomycin, by PAN Biotech (1 mentions)
1.5 ml tube, by Sarstedt (1 mentions)
N garde mycoplasma pcr reagent set, by Euroclone (1 mentions)
Ps 1145, by Merck Group (1 mentions)
Sodium palmitate, by Merck Group (1 mentions)
6 protocols using 60 mm dishes
1
Examining Western Blot Analysis in Human Granulosa Cells
2
Plasma Treatment of HaCaT Keratinocytes
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HaCaT keratinocytes were cultivated in RPMI 1640 cell culture medium containing 8% fetal bovine serum (Sigma-Aldrich, Germany), 2 mM glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin (PAN Biotech, Germany) at 37°C, 95% relative humidity, and 5% CO2 [16 (link)]. Twenty-four hours prior to experiment, 1 × 106 cells were seeded in 60 mm dishes (Sarstedt, Germany). As cold physical plasma source, the kINPen 09 (neoplas tools, Germany) was utilized. This plasma jet consists of a central pin-type electrode that ignited a plasma by applying a voltage of 2–6 kV at a frequency of around 1 MHz. Argon (Air Liquide, France) was used as feed gas (3 standard liters per minute). For all experiments, an indirect treatment regimen was chosen to assure homogeneity of the treatment and it was achieved by exposing 5 ml of RPMI w/ all supplements to the plasma effluent at a distance of 9 mm using an automated xyz-table. The treated liquid was transferred immediately to the prepared cells.
3
Quantifying Cellular Uptake of AuNPs
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U373MG cells were seeded into 60 mm dishes (Sarstedt) at a density of 1 × 105 cells/ml and incubated for 2 days to achieving 90–100% confluency. The culture medium was removed and cells were directly exposed to CAP at 75 kV for corresponding time. The fresh culture medium containing 100 μg/ml AuNPs or 0.1% (w/v) NaN3 was then replaced and incubated for 0.5–48 h at 37 °C or 4 °C as indicated. After treatment, cells were washed thrice with phosphate buffered saline to remove the AuNPs outside cells. Cells were then dissociated and collected from the culture dish using prewarmed (37 °C) 0.25% trypsin solution. The cell suspension was counted using hemocytometer, then transferred to 15 ml tube (Sarstedt) to measure the gold atomic absorbance in AAS. To verify the Au amount of the samples, five-point standard curve were first established using 1–5 ppm standard gold colloid. The concentrations of AuNPs in samples were then calibrated by the standard curve as described elsewhere48 .
4
Adipogenic Differentiation of 3T3-L1 Cells
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The 3T3-L1 cells were seeded in 60-mm dishes (Sarstedt) at a density of 5 × 105 cells in basal medium-BM I (DMEM high glucose, 10% Newborn Calf Serum NCS, antibiotics) for 0–3 days (48 h confluence), as proposed in the protocol from Zebisch et al., (2012). After this time, the differentiation medium (BM I, isobutyl-1-methylxanthine IBMX 0.5 mol, insulin 1 µg/mL, dexametason 0.25 µmol/mL, rosiglitazone 2 µmol/mL) was used for the next 3–5 days (Merck, Darmstadt, Germany). After this time, differentiation medium II was used for the next 5–7 days (BM I, insulin 1 µg/mL), and the basal medium was used for mature adipocytes for 7–14 days. The differentiation process was easily visible. Intracellular lipid droplets appeared at around 7 days and increased in both number and size over the following days. The adipocytes after differentiation were used for the experiment.
The 3T3-L1 cells were incubated with 50 µmol of eicosapentaenoic acid (EPA, dissolved in ethanol) for 48 h and then activated with 10 ng/µL of lipopolysaccharide (LPS isolated from E. coli) or tumor necrosis factor-α (TNF-α, human recombinant) (Sigma-Aldrich, Saint Louis, MO, USA). All experiments were carried out in triplicate, and the number of samples for each group was five (N = 5).
The 3T3-L1 cells were incubated with 50 µmol of eicosapentaenoic acid (EPA, dissolved in ethanol) for 48 h and then activated with 10 ng/µL of lipopolysaccharide (LPS isolated from E. coli) or tumor necrosis factor-α (TNF-α, human recombinant) (Sigma-Aldrich, Saint Louis, MO, USA). All experiments were carried out in triplicate, and the number of samples for each group was five (N = 5).
5
SGBS Preadipocyte Differentiation Protocol
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SGBS (Simpson–Golabi–Behemel syndrome) human preadipocytes were cultured in DMEM-Ham’s F12 (1:1) medium containing 2 mM L-glutamine, 100 I.U./mL penicillin and 100 µg/mL streptomycin, 17 uM pantothenic acid, 33 uM biotin, and 10% of fetal bovine serum (FBS). The cells were seeded in 60 mm dishes (Sarstedt, Nümbrecht, Germany) with 5 mL of culture medium, and maintained at 37 °C and 5% CO2 until they reached complete confluence. The absence of mycoplasma was checked by a polymerase chain reaction (PCR) analysis (N-GARDE Mycoplasma PCR Reagent set, Euroclone, Milan, Italy, EMK090020). When complete confluence was reached (PID0), white adipogenic differentiation was induced using a serum-free medium supplemented with 100 I.U./mL penicillin and 100 µg/mL streptomycin, 33 µM biotin, 17 µM pantothenic acid, 2 µM rosiglitazone, 10 µg/mL human apo-transferrin, 20 nM human insulin, 25 nM dexamethasone, 500 µM 3-isobutyl-1-methylxantine (IBMX), 100 nM cortisol, and 200 pM triiodothyronine. After four days (PID4), the medium was changed and rosiglitazone, dexamethasone, and IBMX were removed. Cells were maintained in the new medium for the remaining 5 days of differentiation until they reached PID9. The differentiation medium was replaced every third day.
6
Hypothalamic Cell Lines and Inflammatory Modulators
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The clonal, embryonic mouse hypothalamic mHypoE-44 and mHypoE-46 cell lines, and the adult mouse hypothalamic mHypoA-NPY/GFP cell line, were generated and culture conditions described previously by our laboratory [16] [17] [18] . Cell lines were in 60 mm dishes (Sarstedt, Montreal, QC)and allowed to reach 70-80% confluence. TNF-α, sodium palmitate and the IKKβ inhibitor, PS1145 were purchased from Sigma. Sterile hypure water was used as the vehicle for TNF-α and sodium palmitate. sodium palmitate was solubilized in sterile hypure water by heating the mixture to 65°C followed by gentle vortexing, and immediately added to culture medium pre-heated to 37°C containing 5% FBS to act as a carrier. The inhibitor PS1145 was prepared in dimethyl sulfoxide (DMSO), and the concentration of DMSO was ~0.01% for treatments. Cells were pre-treated with PS1145 1 hour prior to administration of TNF-α or sodium palmitate.
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