BMDMs from WT and Csf2rb−/− mice were generated and cultured for NMR using procedures described in section 2.2 in complete culture medium containing 10% dialyzed serum (Sigma, St. Louis, USA) and with no HEPES addition. The BMDMs (2x106 cells) were plated in a 100mm tissue culture dish with complete culture medium with dialyzed serum for an additional 48 hours at 37°C prior to collection for NMR analysis. The media collected from cultured cells is termed as conditioned media. Media was also collected at an initial time point (Tinitial or T0 media) before culture. This media is termed “unused media”. Media (1 mL) was centrifuged at 2,000 x g at 4º for 5 minutes and the supernatant (800 μL) was transferred into a new eppendorf tube, frozen in liquid nitrogen, and stored at −80ºC. Just prior to the NMR, samples were thawed on ice and centrifuged 4,000 x g for 5 min at 4°C. The supernatant (600 μL) was aliquoted onto pre-washed 3 kDa spin filters (NANOSEP 3K, Pall Life Sciences), centrifuged 10,000 x g for 90 min at 4°C, and the media filtrate was mixed with NMR buffer up to 600 μL.
Dialyzed serum
Manufactured by Merck Group
Sourced in United States
Dialyzed serum is a specialized laboratory product that undergoes a purification process to remove certain molecules and substances, retaining the desired components. It is commonly used in various experimental and analytical procedures in life science research.
Automatically generated - may contain errors
Lab products found in correlation
13c615n2 lysine, by Cambridge Isotopes (2 mentions)
13c6 15n4 arginine, by Merck Group (2 mentions)
Nanosep 3k, by Pall Corporation (1 mentions)
Rpmi 1640, by Biowest (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Arginine and lysine, by Merck Group (1 mentions)
Quick start bradford assay, by Bio-Rad (1 mentions)
3 protocols using dialyzed serum
1
Preparation of Bone Marrow-Derived Macrophages for NMR Analysis
2
SILAC Labeling of MEF and Kasumi-1 Cells
3
Proteomic analysis of HeLa cells
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HeLa (ATCC: CCL-2) cells were tested for mycoplasma contamination and grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. SILAC media was supplemented with arginine and lysine (SILAC Light) or with heavy isotope-labeled arginine (13C6,15N4-arginine, Sigma) and lysine (13C6,15N2-lysine, Cambridge Isotope Laboratories) (SILAC heavy) in media containing dialyzed serum (Sigma). Cells were cultured at 37 °C in a humidified incubator at 5% CO2. At a confluency of ~90%, cells were washed twice with PBS and lysed in ice-cold modified RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1x mini complete protease inhibitor cocktail (Roche), 10 mM nicotinamide, and 5 μM trichostatin A). Lysates were mixed with 1/10 volume of 5 M NaCl to release chromatin-bound proteins and incubated for 15 min on ice. Subsequently, lysates were homogenized by sonication (6 × 10 sec, 15 W), cleared by centrifugation (20,000 × g, 15 min, 4 °C), and the supernatant precipitated by addition of four volumes of −20 °C acetone. Precipitates were re-dissolved in 8 M guanidine HCl, 50 mM Hepes pH8.5 and protein concentration was determined by Quick-start Bradford assay (Bio-Rad).
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