For western blotting, HEK293T cells were grown in DMEM medium (Sigma-Aldrich, D0819) supplemented with 10% FCS (Sigma-Aldrich, F0392), 1% sodium pyruvate (Sigma-Aldrich, S8636) and 1% penicillin/streptavidin (Sigma-Aldrich, P4333). For immunofluorescence analysis, hTERT RPE-1 and IMCD-3 cells were grown in DMEM/F12, supplemented with 10% FCS (Sigma-Aldrich, F0392), 1% sodium pyruvate (Sigma-Aldrich, S8636) and 1% penicillin/streptavidin (Sigma Aldrich, P4333). IMCD-3 cells stably expressing WASF3 were further supplemented with 400 g/μl hygromycin (Sigma-Aldrich, H0654). All cells were cultured at 37°C and under 5% CO2.
S8636
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
The S8636 is a piece of laboratory equipment manufactured by Merck Group. It serves as a core functional device for various laboratory applications. The product's technical specifications and intended use are not provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (11 mentions)
M7145, by Merck Group (10 mentions)
G7513, by Merck Group (8 mentions)
L glutamine, by Merck Group (7 mentions)
F7524, by Merck Group (7 mentions)
D5671, by Merck Group (4 mentions)
D6171, by Merck Group (3 mentions)
A5955, by Merck Group (3 mentions)
P0781, by Merck Group (3 mentions)
S0615, by Bio&Sell (3 mentions)
G7528, by Merck Group (3 mentions)
G8769, by Merck Group (3 mentions)
M4655, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ab141904, by Abcam (2 mentions)
C2920, by Merck Group (2 mentions)
D6429, by Merck Group (2 mentions)
Sppn980, by NanoComposix (2 mentions)
L6529, by Merck Group (2 mentions)
52 protocols using s8636
1
Western Blotting and Immunofluorescence of Cell Lines
2
Culturing Diverse Cell Lines for Reovirus Propagation
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H1299, T47D, MCF7 and L929 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO2. Adherent L929s were cultured in MEM (M4655, Millipore Sigma) supplemented with 5% FBS (F1051, Millipore Sigma), 1× non-essential amino acids (M7145, Millipore Sigma) and 1mM sodium pyruvate (S8636, Millipore Sigma). Remaining cells were cultured in RPMI (R8758, Millipore Sigma) supplemented similarly to L929 (H1299s in 5% FBS and T47D/MCFs in 10% FBS). L929s cultured in suspension were grown in Joklik’s modified MEM (pH 7.4) (M0518, Millipore Sigma) supplemented with 2g/L sodium bicarbonate (BP328, Fisher Scientific), 1.2g/L HEPES (BP310, Fisher Scientific), 1× non-essential amino acids (M7145, Millipore Sigma) and 1mM sodium pyruvate (S8636, Millipore Sigma). Reovirus serotype type 3 Dearing-PL (T3DPL; Dr. Patrick Lee, Dalhousie University) was propagated in suspension L929 cells from a seed stock to preserve genetic identity, extracted with Vertrel XF (Dymar Chemicals) three times, and purified by ultracentrifugation on cesium chloride gradients, as previously described [72 (link)].
3
Culturing Human Neuroblastoma Cell Lines
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IMR-32 human neuroblastoma cell line was cultured in EMEM medium (M4655, Sigma-Aldrich) supplemented with 10% fetal calf serum (10270106, Gibco), 1% non-essential amino acid solution (M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 50 µg/ml gentamicin (G1397, Sigma-Aldrich). CHP-134 cells were grown in RPMI 1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FCS and 50 µg/ml gentamicin. LAN-1 cells were cultured in EMEM/F-12 (N6658, Sigma-Aldrich) medium diluted in 1:1 ratio, supplemented with 10% FCS, 1% non-essential amino acid solution, 1 mM sodium pyruvate and 50 µg/ml gentamicin, while LAN-5 cells in RPMI 1640 medium supplemented with 20% FCS and 50 µg/ml gentamicin. For preparation of positive controls, IMR-32 and CHP-134 cells were cultured in amino acids-deprived Earle’s Balanced Salt (E2888, Sigma-Aldrich), supplemented with 10% FCS for 24 h. All cell lines were grown at 37 °C in a 5% CO2 atmosphere.
4
Aortic Calcification Quantification
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C57BL/6 mice (8- to 12-week-old male, n = 18) were exterminated by CO2 inhalation and perfused with 5 ml of sterile DPBS. The entire aorta was harvested and cleaned under aseptic conditions and cut into pieces. Aorta rings were randomly divided into three groups and maintained in control, high Pi (2 mmol/L Pi), and high Pi (2 mmol/L Pi) plus DPD (25 μmol/L) conditions, respectively. The culturing medium was DMEM (D6171, Sigma) supplemented with 10% FBS (10,270-106, Gibco, Grand Island, NY, United States), antibiotic–antimycotic solution (A5955, Sigma), sodium pyruvate (S8636, Sigma), l-glutamine (G7513, Sigma), and 2.5 μg/ml amphotericin B (171,375, Millipore). The medium was changed every 2 days. After the 3rd, 5th, and 7th day, the aorta pieces were washed in DPBS, opened longitudinally and decalcified in 25 µL of 0.6 mmol/L HCl overnight. The Ca content was determined by using the QuantiChrom Ca-assay kit, as described earlier. The observer who performed aorta Ca measurements was blinded to the group assignment.
5
Nrl-GFP iPSC Generation and Culture
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We used Nrl-GFP iPSC that were generated [51 (link)] from Nrl-GFP transgenic mice [52 (link)]. Nrl-GFP iPSC was maintained in GMEM (11710–035, Thermo Fisher Scientific) / 10% FBS (555–21245, Biosera)/1 mM sodium pyruvate (S8636, Sigma) / 0.1 mM NEAA (11140–050, Thermo Fisher Scientific) / 0.1 mM 2-mercaptoethanol (2-ME, 137–06862, Wako) supplemented with 1000 U/ml LIF (ESG1107, Esgro), 3 μM CHIR99021 (1677–25, BioVision), and 1 μM PD0325901 (04-0006-10, Stemgent) on 0.1% gelatin-coated dish (G2625, Sigma). Cells were dissociated by 3–4 days using 0.25% trypsin-1 mM EDTA (25200, Thermo Fisher Scientific), and 0.75–1.5 x 105 cells were seeded in a 60-mm dish.
6
Halo-Tag Labeling of NUP96 in U-2 OS Cells
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Human osteosarcoma cells of line U-2 OS, endogenously expressing NUP96 carboxy-terminally tagged with Halo-Tag23 (link) (U-2 OS-CRISPR-NUP96-Halo clone no. 252, CLS GmbH) were grown in McCoy’s medium (16600082, Thermo Fisher Scientific) with 10% (v/v) FBS (S0615, Bio&SELL), 1% (v/v) sodium pyruvate (S8636, Sigma-Aldrich) and penicillin–streptomycin (P0781, Sigma-Aldrich) on coverslips. For fixation, the cells were treated with 8% (w/v) paraformaldehyde (PFA) in PBS at 37 °C for 5 minutes, quenched with 100 mM NH4Cl in PBS for 10 minutes and permeabilized using 0.5% (v/v) Triton X-100 in PBS for 5 minutes. Samples were incubated with Halo-ONB-CP560 (Supplementary Fig. 5 ) at 1 µM in PBS for 1 hour, followed by several washes with PBS for 1 hour. For sample alignment on the microscope, the samples were incubated with primary antibody against NUP153 (ab24700, Abcam), bearing Alexa Fluor 488 (A11001, Invitrogen / Thermo Fisher Scientific, 1:2,000 dilution) for 30 minutes in 2% (w/v) BSA in PBS. Finally, the cells were washed with PBS.
7
Caco-2 Cell Culture Maintenance Protocol
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Human epithelial cell line Caco-2 was grown in monolayers and maintained at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity. The cells were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Sigma® St. Louis, MI, USA, F4135), 1% sodium pyruvate (Sigma® S8636), 1% transferrin (Sigma® T8158), 1% antibiotic/antimycotic solution (100 units/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL of amphotericin B) (Sigma® A5955), and 1% L-glutamine (Biowest®, Nuaillé, France, X0551-100). The cell culture medium was changed every 2 to 3 days and the cells were kept in exponential growth in T75 flasks. At 90% confluence, a subculture was performed with the harvested cells by trypsinization (0.25%, w/v, trypsin−EDTA4−Na), with a split ratio of 1:3.
8
Seahorse XF Mitochondrial Respiration Assay
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Assays were performed using a Seahorse XF 96 analyzers (Agilent, USA) according to the manufacturer’s instructions. Concisely, 8 × 103 cells/well were seeded in a 96-well XF cell culture microplate and incubated overnight in the recommended growth medium before the assay. The sensor cartridge was hydrated in a Seahorse XF Calibrant at 37 °C in a CO2-free incubator overnight. Subsequently, the medium was replaced with CO2-free low-buffered assay medium containing 2 mM L-glutamine (Lonza, 17-605E), 1 mM pyruvate (Sigma, S8636) and 25 mM glucose (Sigma, G7528). The oxygen-consumption rate (OCR) was measured at baseline and following sequential addition of 2 µM oligomycin (Abcam, ab141829), 1 µM FCCP (Sigma, C2920), 5 µM rotenone (Sigma, C2920), and 5 µM antimycin A (Abcam, ab141904). We used the total number of cells per well to normalize the OCR data.
9
Nitrite Quantification in Cdk5-Deficient Macrophages
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Bone marrow derived macrophages were isolated from Cdk5flox and Cdk5LysMCre mice and grown until day 6. Afterwards, cells were seeded in a 96-well plate (150'000 cells/well) with DMEM media without phenolred (D1145, sigma) supplemented with 10% fetal bovine serum (FBS, F7524, sigma), 20 ng/ml M-CSF (R&D system), 1% Penicillin/Streptomycin (P0781, sigma), 1% L-Glutamine (G7513, sigma), 1% Sodium Pyruvate (S8636, sigma), and incubated at 37°C and 5% CO2. At day 7, BMDMs were treated for the indicated time points. Supernatant was collected after 48 h, centrifuged (13'000 rpm, 5 min) and nitrite was measured as a stable metabolite of NO with Griess reagent (Molecular Probes; G7921) according to the manufacturer's protocol.
10
Culturing Canine Tumor Cell Lines
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Two canine tumor cell lines (K9STS, mesenchymal tumor; K9MM2, round cell tumor) derived from primary tumors were kindly provided by Professor Dr. Rohrer Bley. K9STS cells were cultured at 37 °C under 5% CO2 in a humidified atmosphere in RPMI 1640 medium (R8758, Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (F7524, Sigma Aldrich, St. Louis, MO, USA), 1% HEPES (15630-056, Gibco, Thermo Scientific, Rockford, IL, USA), 1% l -glutamine (G7513, Sigma Aldrich, St. Louis, MO, USA), 1% MEM non-essential amino acid (NEAA) (11140-050, Gibco, Thermo Scientific, Rockford, IL, USA), 1% sodium pyruvate (S8636, Sigma Aldrich, St. Louis, MO, USA), and 1% penicillin-streptomycin (P0781, Sigma Aldrich, St. Louis, MO, USA). K9MM2 cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (D6429, Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FCS, 1% HEPES, and 1% penicillin-streptomycin at 37 °C under 5% CO2 in a humidified atmosphere. Cells were regularly passaged every second day and used for experiments in the exponential growth phase. Cell lines were tested negative for mycoplasma contamination.
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