Dialyzed fbs

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Dialyzed Fetal Bovine Serum (Dialyzed FBS) is a cell culture reagent. It is processed to remove low molecular weight components, such as hormones, growth factors, and other small molecules, while retaining larger proteins and other macromolecules. Dialyzed FBS is commonly used in cell culture applications where the presence of these small molecules may interfere with experimental conditions or the behavior of the cells being studied.

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Dialyzed (15 citations)

100 protocols using dialyzed fbs

SILAC Isotopic Labeling for Quantitative Proteomics

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The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within four passages. Cells were then plated at a density of 500,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L and 15% dialyzed FBS (Thermo Scientific). Cells were collected after 0, 1, 2, and 3 d of labeling and washed with PBS, and cell pellets were frozen before further analysis. Eight days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L and 15% dialyzed FBS (Thermo Scientific). Cells were collected after 0, 2, 4, and 6 d of labeling and washed with PBS, and cell pellets were frozen before further analysis.

Zhang T., Wolfe C., Pierle A., Welle K.A., Hryhorenko J.R, & Ghaemmaghami S. (2017). Proteome-wide modulation of degradation dynamics in response to growth arrest. Proceedings of the National Academy of Sciences of the United States of America, 114(48), E10329-E10338.

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Stable Isotopic Labeling in Cell Culture

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Before isotopic labeling, cultures were grown to confluency. Once cells ceased cell division and were contact inhibited, they were maintained in a quiescent state for 4 days. Subsequently, cells were acclimated to the labeling media (EMEM) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin for 4 days. After 4 additional days in the adaptation media, cultures were introduced to the minimum essential medium for stable isotopic labeling in cell culture (SILAC) (Thermo Scientific) supplemented with L-arginine:HCl (¹³C6, 99%) and L-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.13 g/l and 0.0904 g/l, respectively, 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. After 0, 2, 4, and 6 days of labeling, cells were harvested and washed with PBS, and cell pellets were frozen before further analysis.

Swovick K., Firsanov D., Welle K.A., Hryhorenko J.R., Wise JP S.r., George C., Sformo T.L., Seluanov A., Gorbunova V, & Ghaemmaghami S. (2021). Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands. Molecular & Cellular Proteomics : MCP, 20, 100041.

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Isotopic Labeling Procedure for Proteome Analysis

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The isotopic labeling procedure was similar to that previously described by Zhang et al.^{35 (link)} The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within five passages. Cells were then plated at a density of 1,000,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L, 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. For whole proteome analysis, cells were collected after 0, 1, 2, 3, and 5 days of labeling and washed with PBS. For analysis of isolated PrP^Sc aggregates, cells were collected after 0, 1, 2, 3, and 4 days of labeling and washed with PBS. All cell pellets were frozen before further analysis.

Hutti C.R., Welle K.A., Hryhorenko J.R, & Ghaemmaghami S. (2020). Global analysis of protein degradation in prion infected cells. Scientific Reports, 10, 10800.

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Stable Isotope Labeling of Quiescent Cells

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Prior to isotopic labeling, cultures were grown to 100% confluency. Once cells ceased cell division and were contact inhibited, they were maintained in a quiescent state for 4 days. Subsequently, cells were acclimated to the labeling media (EMEM) supplemented with 15% dialyzed FBS (Thermo Scientific), 100U/ml penicillin, and 100 U/ml streptomycin for 4 days. After 4 additional days in adaptation media, the cultures were introduced to MEM media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl ( 13 C6, 99%) and L-lysine:2HCl ( 13 C6, 99%;Cambridge Isotope Laboratories) at concentrations of 0.13 g/l and 0.0904 g/l respectively, 15% dialyzed FBS (Thermo Scientific), 100U/ml penicillin, and 100 U/ml streptomycin. After 0, 2, 4, and 6 days of labeling, cells were harvested, washed with PBS, and pellets were frozen before further analysis.

Swovick K., Firsanov D., Welle K.A., Hryhorenko J.R., Wise J.P., George C., Sformo T.L., Seluanov A., Gorbunova V., & Ghaemmaghami S. (2020). Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands.

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Mitochondrial Transfer to Revive ρ0 Cells

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Example 4

Mitochondria were harvested from DsRed HEK293T, LP351 PBMC, or LP298 PBMC using a Qproteome Mitochondria Isolation Kit (Qiagen, Cat. #37612) following manufacturer's protocol. Mitochondrial pellets were resuspended in PBS at a concentration ˜1 mg total protein/mL. Mitochondrial suspensions were delivered into ρ0 fibroblast cells using the device described herein. Transferred fibroblasts were cultured in complete media with 50 μg/mL uridine for 4 days following mitochondria delivery. On day 5, cells were shifted to uridine-free complete media prepared with 10% dialyzed FBS (Life Technologies, Cat. #26400-044). On day 8, cells were shifted to glucose-free, galactose-containing medium (DMEM without glucose (Gibco, Cat. #11966025) supplemented with 10% dialyzed FBS and 4.5 g/l galactose). Colonies emerged ˜10 d post-delivery and cells were shifted back to uridine-free medium before colonies were counted by microscopy or isolated using cloning rings.

US11639510B2. Devices and methods for mitochondria replacement and for generating cellular therapeutics (2023-05-02). NanoCav, LLC [US]. Inventors: Ting-Hsiang Sherry Wu [US], Artin Mehrabi [US], Jon Thomas Van Lew [US].

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SILAC Labeling Efficiency Validation

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PANC-1cells were cultured in SILAC DMEM medium (Welgene, Daegu, Korea) with dialyzed FBS (Gibco, MA, USA) containing heavy 0.798 mM lysine and 0.398 mM arginine. Heavy lysine (1G: CLM-265-H-1) and arginine (1G: CNLM-291-H-1) were purchased from Cambridge Isotope Laboratories (CIL, Andover, MA, USA). PANC-1R cells were grown in light SILAC growth medium (DMEM, Capricorn Scientific GmbH, Germany) with dialyzed FBS (Gibco, MA, USA). All cells were maintained at 37 °C in humidified air containing 5% CO₂. To validate labelling efficiency for full incorporation of heavy amino acid labels in all proteins, cells were cultured for seven passages and checked reached > 95% by LC-MS/MS analysis.

Kim Y.E., Kim E.K., Song M.J., Kim T.Y., Jang H.H, & Kang D. (2021). SILAC-Based Quantitative Proteomic Analysis of Oxaliplatin-Resistant Pancreatic Cancer Cells. Cancers, 13(4), 724.

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Rapamycin-Induced Phosphotyrosine Profiling in HeLa Cells

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Split-Abl engineered Hela cells (100,000/well) were plated and grown to confluency on acid-washed glass coverslips (Fisher) for 24 hours. The following day, cells were treated for one hour with 10 nM Rapamycin or DMSO in complete DMEM media. Cells were fixed and permeabilized with 4% PFA in PBS for 15 minutes. Coverslips were washed with PBS + 0.1% Triton X-100 (PBS-TX) and then incubated with mouse anti-phospho-Tyr-100 (CST, 1:200) diluted in PBS-TX + 10% dialyzed FBS (Thermo Scientific) for 30 minutes at 37 °C. Coverslips were washed once with PBS for five minutes, washed once with PBS-TX for five minutes, then incubated with 1:100 Alexa Fluor 488 anti-mouse, DAPI 33342 for 30 minutes at 37 °C. Coverslips were mounted using SlowFade Gold (Molecular Probes) and visualized on a Ziess Axioobserver microscope.

Diaz J.E., Morgan C.W., Minogue C.E., Hebert A.S., Coon J.J, & Wells J.A. (2017). A Split-Abl Kinase for Direct Activation in Cells. Cell chemical biology, 24(10), 1250-1258.e4.

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SILAC Labeling of U2OS Cell Lines

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U2OS cells (wild-type and knockout cell lines) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin/streptomycin (100U/ml) at 37°C, 5% CO2. Cells were regularly tested for Mycoplasma by PCR-based detection analysis and discarded if positive.
For SILAC labeling (Ong et al., 2002 (link)), U2OS cells (wild-type and knockout cell lines) were grown in medium containing unlabelled L-lysine as the light condition, or isotopically labeled L-lysine (¹³C6,¹⁵N2) as the heavy condition. Both light and heavy DMEM were supplemented with 10% dialyzed FBS (Thermo Scientific). Cells were cultured for more than 7 generations to achieve complete labeling. Incorporation efficiency (> 99%) was determined by MS.

Bonfiglio J.J., Fontana P., Zhang Q., Colby T., Gibbs-Seymour I., Atanassov I., Bartlett E., Zaja R., Ahel I, & Matic I. (2017). Serine ADP-Ribosylation Depends on HPF1. Molecular Cell, 65(5), 932-940.e6.

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SILAC Labeling of U2OS Osteosarcoma Cells

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Human U2OS osteosarcoma cells and HEK293T cells (293T) were acquired from ATCC. Chicken bursal lymphoma DT40 cells were a generous gift from Prof. William Earnshaw (University of Edinburgh). U2OS and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin/streptomycin (100U/ml) at 37 °C, 5% CO₂. DT40 cells were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum, 1% chicken serum, penicillin/streptomycin (100U/ml) at 37 °C, 5% CO₂. All cell lines were regularly tested for Mycoplasma by PCR-based detection analysis and discarded if positive.
For SILAC labeling18 (link), U2OS cells were grown in medium containing unlabeled L-lysine (#L8662, Sigma-Aldrich) as the light condition, or isotopically labeled L-lysine (¹³C6,¹⁵N2, # 608041, Sigma-Aldrich) as the heavy condition. Both light and heavy DMEM were supplemented with 10% dialyzed FBS (Thermo Scientific). Cells were cultured for more than 7 generations to achieve complete labeling. Incorporation efficiency (>99%) was determined by MS.

Leidecker O., Bonfiglio J.J., Colby T., Zhang Q., Atanassov I., Zaja R., Palazzo L., Stockum A., Ahel I, & Matic I. (2016). Serine is a new target residue for endogenous ADP-ribosylation on histones. Nature chemical biology, 12(12), 998-1000.

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Galactose Culture of HeLa Cells

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HeLa cells were obtained from RIKEN BioResource Research Center (BRC) and cultured in standard Dulbecco's modified Eagle' medium (DMEM high-glucose 4.5 g/l, Nacalai) with 10% FBS (Biosera), 1 × glutamine (Gibco), and 1 × Penicillin–Streptomycin (Gibco) at 37 °C humidified incubators with 5% CO₂ supplied. For cell growth experiment under the respiratory condition, we used modified Dulbecco's modified Eagle' medium (No Glucose, Nacalai) with the addition of 4.5 g/l D(+)-galactose (Wako), 10% dialyzed FBS (Thermo Fisher Scientific), and 1 × Penicillin–Streptomycin (Gibco). The medium was changed routinely every 48 h. For the cell counting experiment, 0.5 million cells were seeded on a 10-cm dish for cell counting experiment. Cell counts started the next day after seeding for five consecutive days.

Fang Q., Kimura Y., Shimazu T., Suzuki T., Yamada A., Dohmae N., Iwasaki S, & Shinkai Y. (2022). Mammalian HEMK1 methylates glutamine residue of the GGQ motif of mitochondrial release factors. Scientific Reports, 12, 4104.

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