Mt35010cv

Manufactured by Thermo Fisher Scientific

The MT35010CV is a laboratory centrifuge designed for a variety of applications. It features a maximum speed of 10,000 RPM and a maximum RCF of 12,100 x g. The centrifuge has a compact footprint and can accommodate a range of rotor sizes and sample volumes.

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5 protocols using mt35010cv

Culturing U-251 MG and GBM43 Glioblastoma Cells

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U-251 MG (University of California, Berkeley Tissue Culture Facility, which sources from ATCC) GBM cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (Corning, MT 35-010-CV), 1% (vol/vol) penicillin-streptomycin (Thermo Fisher Scientific), 1% (vol/vol) MEM non-essential amino acids (Thermo Fisher Scientific), and 1% (vol/vol) sodium pyruvate (Thermo Fisher Scientific). GBM43 cells (Mayo clinic) were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (Corning, MT 35-010-CV), 1% (vol/vol) penicillin-streptomycin (Thermo Fisher Scientific) and 1% (vol/vol) Glutamax (Thermo Fisher Scientific, 35-050-061). Cells were harvested using 0.25% Trypsin-EDTA (Thermo Fisher Scientific) and passaged less than 30 times. All cells were screened on a bimonthly basis for mycoplasma and validated every six months by Short Tandem Repeat (STR) analysis at the University of California Cell Culture Facility.

Garcia J.H., Akins E.A., Jain S., Wolf K.J., Zhang J., Choudhary N., Lad M., Shukla P., Gill S., Carson W., Carette L., Zheng A., Kumar S, & Aghi M.K. (2023). Multi-omic screening of invasive GBM cells in engineered biomaterials and patient biopsies reveals targetable transsulfuration pathway alterations. bioRxiv.

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Cell Culture and Differentiation Protocols

Cited 1 time

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MDA-MB-231 cells were cultured in DMEM growth medium (Gibco, 11039-021) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning, MT35010CV) and 1× Anti-Anti (Thermo Fisher Scientific, 15240112) at 37°C with 5% CO₂. Undifferentiated HC11 cells were cultured in growing medium (RPMI-1640; Thermo Fisher Scientific, 72400047), supplemented with 10% FBS, 5 µg/ml insulin (Millipore-Sigma, I6634), 10 ng/ml epidermal growth factor (EGF; Preprotech, AF-100-15), 1× Anti-Anti at 37°C with 5% CO₂. Competent HC11 cells were primed for differentiation by culturing them in priming medium [RPMI-1640 supplemented with 5% charcoal-stripped-FBS (Equitech Bio, SFBM31), 5 µg/ml insulin, 1 µM dexamethasone (Millipore-Sigma, D4902-1G) and 1× Anti-Anti] for 18 h at 37°C with 5% CO₂. To induce differentiation, primed HC11 cells were cultured in DIP Medium [RPMI-1640, supplemented with 10% FBS, 5 µg/ml insulin, 1 µM dexamethasone, 1× Anti-Anti and 3 µg/ml Prolactin (NHPP, oPRL-21)] at 37°C with 5% CO₂. Primary cells were harvested from 8- to 12-week-old mice and cultured as previously described (Macias et al., 2011 (link); Rubio et al., 2020 (link)).

Cazares O., Chatterjee S., Lee P., Strietzel C., Bubolz J.W., Harburg G., Howard J., Katzman S., Sanford J, & Hinck L. (2021). Alveolar progenitor differentiation and lactation depends on paracrine inhibition of notch via ROBO1/CTNNB1/JAG1. Development (Cambridge, England), 148(21), dev199940.

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Cell Line Cultivation and Reagent Acquisition

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All chemicals were purchased from Sigma Aldrich unless otherwise noted in Supplemental Figure 1. The ASZ and BSZ cell lines were obtained from Professor Robert Holmgren at Northwestern University. FBS, M154 media, DPBS, and NucBlue stain were obtained from ThermoFisher (MT35010CV, M154CF500, 14190250, and R37605, respectively). Trypsin was obtained from Gibco^™ (TrypLE, 12563011) Pen/Strep was obtained from Sigma Aldrich (P0781). All DNA sequences were obtained from IDT with standard desalting and HPLC purification.

Dukes M.W., Bajema E.A., Whittemore T.J., Holmgren R.A, & Meade T.J. (2022). Delivery of Targeted Co(III)-DNA Inhibitors of Gli Proteins to Disrupt Hedgehog Signaling. Bioconjugate chemistry, 33(4), 643-653.

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Cell Lines and Zebrafish Maintenance

Cited 2 times

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HEK293FT cells (gift of P. Beachy, Stanford University), IMCD3 Flp-in cells (gift of P. Jackson, Stanford University), Sf9 cells (gift of K.C. Garcia, Stanford University), Smo^−/− MEFs (gift of P. Beachy, Stanford University), and HEK293-Freestyle cells (gift of R. Majeti, Stanford University) were grown as previously described^{29 (link)}. NIH3T3 Flp-in cells (gift of R. Rohatgi, Stanford University) were grown according to the manufacturer’s instructions in DMEM (MT10013CM, Fisher Scientific) with 10%FBS (MT35010CV, Fisher Scientific). Stably transfected HEK293 Flp-in T-rex cells (gift of D. Julius, UCSF) were constructed and maintained as previously described^{11 (link)}. IMCD3 stable lines coexpressing FLAG-SMO + PKAC-mNG or FLAG-SMO + β2ARNb80-mNG were previously described, and the stable line expressing FLAG-SMO(WRR) + PKACmNG was produced as previously described. Zebrafish were maintained as previously described^{29 (link)}.

Happ J.T., Arveseth C.D., Bruystens J., Bertinetti D., Nelson I.B., Olivieri C., Zhang J., Hedeen D.S., Zhu J.F., Capener J.L., Bröckel J.W., Vu L., King C.C., Ruiz-Perez V.L., Ge X., Veglia G., Herberg F.W., Taylor S.S, & Myers B.R. (2022). A PKA Inhibitor Motif within SMOOTHENED Controls Hedgehog Signal Transduction. Nature structural & molecular biology, 29(10), 990-999.

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Isolation and Culture of Primary Human Corneal Fibroblasts

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PHCFs were isolated from 74-year-old human donor corneas (obtained through the National Disease Research Interchange) following the procedure established by Bueno et al. (2009) 33 (link) . Briefly, donor corneas were gently scraped of their epithelium and endothelium to ensure only stromal cells were isolated. Then, the corneas were cut into 2x2 mm pieces and sterilized by soaking in 1X Gibco phosphate buffered saline (PBS) (Fisher Scientific, 10-010-023) containing 1% HyClone penicillin streptomycin 100X solution (Fisher Scientific, SV30010) and 0.1% amphotericin B 250 μg/mL solution (Sigma Aldrich, 1397-89-3). Each explant was adhered to the center of a 6well culture plate (Corning, 3516) and PHCF were cultured using the reagents and techniques established by Siadat et al. (2021) 57 (link) . Complete media, comprising Corning DMEM with Lglutamine and 4.5 g/L glucose without sodium pyruvate (Fisher Scientific, MT10017CV), 1% penicillin streptomycin, 0.1% amphotericin B, and 10% Corning regular fetal bovine serum (Fisher Scientific, MT35010CV), was gently added to the well. Cells were incubated at 37 ℃ with 5% CO2. A half media exchange was performed every 3-5 days until the PHCF migrated off the explant. Once confluent, the explants were discarded and PHCF were expanded and frozen for future use.

Silverman A.A., Olszewski J.D., Siadat S.M., & Ruberti J.W. (2021). Mechanical Causation of Biological Structure: Productive Pulls Produce Persistent Filaments in a Human Fibroblast Model of Matrix Development.

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