Mouse embryonic fibroblasts (MEFs) were seeded at 300,000 cells per well in 6-well plates. For [13C6]-glucose labeling, cells were incubated for 12 h in DMEM media (US Biological; Swampscott, MA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 25 mM [13C6]-glucose, ± TGFβ. For [13C5]-glutamine labeling, cells were incubated ± TGFβ for 12 h. During the last hour, media was changed to DMEM containing 1 mM pyruvate, 4 mM [13C5]-glutamine, and 25 mM glucose (99% purity of stable isotope tracers, microbiological and pyrogen tested: Cambridge Isotope Laboratories, Inc., Tewksbury, MA). Cell reactions were then quenched in cold acetonitrile and extracted in acetonitrile:water:chloroform (v/v/v, 2:1.5:1), as described previously [35 (link)–38 (link)], to obtain the polar, nonpolar, and insoluble proteinaceous fractions. The nonpolar (lipid) layer was collected, dried under a stream of nitrogen gas, and reconstituted in 0.1 mL of chloroform:methanol:butylated hydroxytoluene (2:1 + 1 mM) mixture and stored at −80 °C for future analysis. The polar fraction was lyophilized using a Freezone 2.5 L −84 °C benchtop freeze dryer (Labconco, Kansas City, CO, USA). The dried sample was reconstituted in 100 μL 20% acetonitrile and used for LC-MS analysis.
Freezone 2.5 l 84 c benchtop freeze dryer
Manufactured by Labconco
Sourced in United States
The Freezone 2.5 L -84 °C benchtop freeze dryer is a compact laboratory equipment designed for freeze drying applications. It features a 2.5 L freeze drying chamber capable of reaching temperatures as low as -84 °C.
Automatically generated - may contain errors
Lab products found in correlation
Dmem media, by US Biological (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Pyruvate, by Cambridge Isotopes (1 mentions)
Glutamine, by Cambridge Isotopes (1 mentions)
13c6 glucose, by Cambridge Isotopes (1 mentions)
Spectrum 100, by PerkinElmer (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
13c6 glucose, by Merck Group (1 mentions)
Whatman no 2 filter paper, by Cytiva (1 mentions)
5 protocols using freezone 2.5 l 84 c benchtop freeze dryer
1
Isotopic Tracing of Metabolic Flux in Mouse Embryonic Fibroblasts
2
Isotopic Labeling and Metabolomic Analysis
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NRCMs were incubated in 6-well plates for 5 min or 4–18 h in DMEM media (US Biological; Swampscott, MA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 25 mM [13C6]-glucose (99% purity, microbiological and pyrogen tested; Cambridge Isotope Laboratories, Inc., Tewksbury, MA). Additionally, for compound/inhibitor experiments, FCCP, KA, Oligo, Rot, or 2DG were added to this [13C6]-glucose containing media. After the specified isotope labeling time point, cell reactions were quenched in cold acetonitrile, and extracted in acetonitrile:water:chloroform (v/v/v, 2:1.5:1), as described previously31 (link),32 (link),60 (link),61 (link), to obtain the polar, nonpolar, and insoluble proteinaceous fractions. The nonpolar (lipid) layer was collected, dried under a stream of nitrogen gas, and reconstituted in 0.1 ml of chloroform:methanol:butylated hydroxytoluene (2:1 + 1 mM) mixture and stored at −80 °C for future analysis. The polar fraction was lyophilized using a Freezone 2.5 L −84 °C benchtop freeze dryer (Labconco, Kansas City, CO, USA). The dried sample was reconstituted in 100 μl 20% acetonitrile and used for LC-MS analysis.
3
FTIR Analysis of CNC Forms
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FTIR
spectroscopy (Spectrum 100, PerkinElmer, USA) was used to distinguish
sCNCs, DACNCs, and cysCNCs. A small portion of each form of the CNC
gels was thoroughly washed and freeze-dried with a FreeZone 2.5 L
−84C Benchtop Freeze-Dryer (Labconco Corporation) to obtain
the required solid samples. The absorbance of the IR signals (600–4000
cm–1) was normalized using a band located at 1030
cm–1 for all the CNC samples and compared with cystamine
dihydrochloride.
spectroscopy (Spectrum 100, PerkinElmer, USA) was used to distinguish
sCNCs, DACNCs, and cysCNCs. A small portion of each form of the CNC
gels was thoroughly washed and freeze-dried with a FreeZone 2.5 L
−84C Benchtop Freeze-Dryer (Labconco Corporation) to obtain
the required solid samples. The absorbance of the IR signals (600–4000
cm–1) was normalized using a band located at 1030
cm–1 for all the CNC samples and compared with cystamine
dihydrochloride.
4
Tracing Glucose Metabolism in iPSC-CMs
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Human iPSC-CMs cells were plated at 300,000 cells per well in a 6-well plate then transduced with adenovirus as described above. After 24 h, the cells were incubated in medium containing 11 mM 13C6-glucose (Sigma Aldrich 389374) for 3, 8, or 16 h in RPMI 1640 media (Gibco 11879–020) for 16 h. Media in both treatments was supplemented with B27 supplement (ThermoFisher 17504044) and penicillin/streptomycin (ThermoFisher 15070063). Metabolism was quenched and metabolites were extracted in an ice-cold acetonitrile:water:chloroform solution at a final ratio of 2:1.5:1 [35 ,36 (link)]. Polar fractions were lyophilized using a Freezone 2.5L −84 °C benchtop freeze dryer (Labconco, Kansas City, CO, USA). The dried sample was reconstituted in 100 μl 20% acetonitrile and used for LC-MS analysis.
5
Microalgae Biomass Extraction Protocol
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Prior to biomass harvesting, 30-day-old microalgae batch cultures were spotted onto Lysogeny Broth (LB) agar plates (1.5% w/v, Bacto agar) and incubated at 37 °C in a New Brunswick™ Innova incubator (Eppendorf, Hauppauge, NY, USA). After 24 h, agar plates were visually inspected for microbial contamination. Only cultures that exhibited no signs of contamination were considered for further studies. Microalgae cultures were harvested by centrifugation (4000 rpm, 15 min). The biomass was flash-frozen in liquid nitrogen and freeze-dried using a FreeZone 2.5 L, −84 °C Benchtop Freeze Dryer (Labconco, Kansas City, MO, USA) under 0.15 mbar vacuum pressure and a collector temperature of −80 °C for 48 h. From the resulting dried biomass, aliquots of 250 mg were transferred to glass vials and extracted separately with 8 mL of four different solvents (hexane, methanol, chloroform, and ethanol). The biomass with each solvent was sonicated for 30 min to favor tissue disruption and then filtered with Whatman No. 2 filter paper. The filtered extracts were dried out in the fume hood for 30 h. When completely dried, the samples were resuspended in 250 µL of the corresponding extraction solvent and used in the different assays.
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