The Human adult cardiac fibroblast (HCF) were obtained from Promocell (C-12375) and they were expanded according to the protocol [24 ]. Briefly, a T175 cell culture flask (Greiner) was incubated (at 37°C and 5% CO2) with 12 mL of FGM-3 (Promocell, C-23130) for 30 minutes. The cryovial containing the HCF was thawed in a water bath at 37°C. Then, the cells were transferred from the cryovial to a cell culture flask containing the FGM-3 with an additional 18 ml of FGM-3. Subsequently, the culture flask was placed inside the incubator. Refreshments were done every 48 hours. When the cells reached 70–90% confluency they were passaged to a new flask; this process was repeated until reaching 11 passages. At the last passage, the HCF were frozen at a final concentration of 150 x 103cells/ 0.5 mL in freezing medium. The freezing medium consists of 50% KOSR (Thermo Fisher, 10828028), 40% FGM-3, 10% DMSO (Sigma-Aldrich, D2650) and 0.5% Revitacell (Thermo Fisher, A2644501).
D2650
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D2650 is a laboratory equipment product from Thermo Fisher Scientific. It is a spectroscopic instrument that can be used for various analytical applications. The core function of the D2650 is to perform spectroscopic analysis, but no further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fgm 3, by PromoCell (1 mentions)
Revitacell, by Thermo Fisher Scientific (1 mentions)
C 12375, by PromoCell (1 mentions)
T175 cell culture flask, by Greiner (1 mentions)
Fgm 3, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Stem cellbanker, by AMS Biotechnology (1 mentions)
Cryostor 10, by BioLife Solutions (1 mentions)
4 protocols using d2650
1
Expansion and Cryopreservation of Human Cardiac Fibroblasts
2
Isolation and Cryopreservation of Omicron-Infected Splenocytes
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Splenocytes were collected from the Omicron B.1.1.529 infected mice 21 days post-infection. A spleen was removed from the animal and collected in media (90% RPMI 1640 (Gibco Cat. 11875093, Waltham, MA, USA) + 10% FBS (VWR Cat. 89510-188, Radnor, PA, USA)). After, the spleen was smashed through a 70 µm mesh strainer (Corning cat. 352350, Corning, NY, USA), and subsequently, red-blood-cell lysed according to manufacturer’s instructions (Invitrogen cat. 00430054, Waltham, MA, USA). Isolated splenocytes were preserved in cryomedia (45% RPMI 1640 + 45% FBS + 10% DMSO (Sigma cat. D2650, St. Louis, MO, USA)), frozen in Mr. Frosty freezing containers (ThermoFisher cat. 5100-0001, Waltham, MA, USA) at ≤−65 °C, then stored in the vapor phase of liquid nitrogen until use.
3
Cryopreservation of Cultured Cells
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Flasks were washed with pre-warmed 37°C PBS without Ca 2+ and Mg 2+ and incubated for 15 min at 37°C, 5% CO 2 /21% O 2 with TrypLE Select. TrypLE Select was deactivated using DTI, and cells were scraped from the flask surface using a cell scraper (VWR, 734-2604). After centrifugation at 300g for 3 min, cells were resuspended in NSÀ/À with P/S and filtered with a 40-mm cell strainer before counting. Cells were frozen at a standard cell density of 5 £ 10 5 À5 £ 10 6 cells/vial according to the manufacturing instructions. Cells were again centrifuged and resuspended in an adequate volume in cryopreservation solutions (STEM-CELLBANKER [11897; Amsbio, Abingdon, UK], CryoStor 10 [210374; BioLife Solutions, Bothell, WA, USA] and 10% DSMO [D2650; Thermo Fisher Scientific]) in NSÀ/À and transferred to a cryotube, placed in a CoolCell that allows gradual temperature reduction, frozen in a À80°C freezer for a day and then transferred to a nitrogen tank.
4
BV2 Microglial Cell Culture
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