Silac rpmi 1640

Manufactured by Thermo Fisher Scientific

Sourced in United States

SILAC RPMI 1640 is a cell culture medium formulation designed for stable isotope labeling by amino acids in cell culture (SILAC) experiments. It contains the necessary nutrients and amino acids for cell growth and protein labeling.

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RPMI 1640 (15891 citations) SILAC (13 citations) SILAC RPMI (5 citations) RPMI 1640 medium for SILAC (4 citations) SILAC RPMI 1640 Flex Media (2 citations) SILAC RPMI 1640 medium (2 citations)

6 protocols using silac rpmi 1640

Culturing CD19-CAR T Cells and Raji B Cells

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CD19-CAR T cells and Raji B cells were initially cultured in RPMI 1640 supplemented with 10% (v/v) FBS, 2mM L-glutamine, 100μ/mL penicillin, and 100 μg/mL Streptomycin and maintained under standard conditions (5% CO₂, 37 °C). After 12 days in culture, Raji B cells were collected and washed twice with SILAC RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) before reconstitution in SILAC RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) containing 0.38 mM ¹³C₆, ¹⁵N₄ Arginine, 0.22 mM ¹³C₆, ¹⁵N₂ Lysine (Cambridge Isotope Laboratories, Andover, MA), 10% dialyzed FBS (Gibco Life Technologies, Guilford, CT), 2 mM L-glutamine, 100 U/mL Penicillin, and 100 μg/mL Streptomycin (Cytiva, Malborough, MA) and maintained for a total of 8 doublings under standard conditions (5% CO₂, 37 °C). During this time, CD19-CAR T cells were expanded and maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL Penicillin, and 100 μg/mL Streptomycin.

Griffith A.A., Callahan K.P., King N.G., Xiao Q., Su X, & Salomon A.R. (2022). SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells. Journal of proteome research, 21(2), 395-409.

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Bone Marrow Dendritic Cell Culture

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BMDC-RPMI: 10% fetal bovine serum (FBS, HyClone), 1% L-glutamine (Gibco), 1% Penicillin/Streptomycin (P/S, Gibco), 55μM 2-ME (Sigma), and 10μg/mL mouse GM-CSF (R&D Systems) in RPMI-1640 (Corning). Complete (C)-DMEM: 10% bovine calf serum (BCS, HyClone,) and 1% P/S in standard DMEM (Corning). L-ARG-free SILAC RPMI: 10% dialyzed FBS (Corning), 1% P/S, 219μM L-lysine-HCl (Sigma), and 55μM 2-ME in SILAC RPMI-1640 (Thermo Scientific).

Crowther R.R., Schmidt S.M., Lange S.M., McKell M.C., Robillard M.C., Zhao J., Haffey W.D., Wyder M.A., Greis K.D., Setchell K.D, & Qualls J.E. (2022). L-arginine transfer from antigen presenting cells sustains CD4+ T cell viability and proliferation. Journal of immunology (Baltimore, Md. : 1950), 208(4), 793-798.

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CD44v9 Regulation of EMT in ESCC

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Human ESCC cell lines (TE1, 3, 5, 6, 8, 11, 12, 13) were cultured in SILAC RPMI‐1640 (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Cromwell, CT, USA), at 37°C in an atmosphere containing 5% CO₂.
A CD44v9‐specific siRNA28 was synthesized by Thermo Fisher Scientific. Among cell lines tested the expression of CD44v9 was highest in TE6 cells, which were therefore selected for further study. TE6 cells were seeded in a 6‐well plate (0.25 × 10⁶ cells per well) and reverse transfected with 30 nmol of CD44v9 siRNA in the presence of Lipofectamine RNAimax reagent (Thermo Fisher Scientific) for 72 hour, according to the procedure provided by the manufacturer. A duplex Stealth RNAi (siCONT) was used as a nontargeting siRNA.
To induce EMT, TE6 cells were seeded in a 6‐well plate (0.25 × 10⁶ cells per well) and treated with TGF‐β1 (final concentration of 20 ng/mL) (Invitrogen, Carlsbad, CA, USA) for 72 hour.

Taniguchi D., Saeki H., Nakashima Y., Kudou K., Nakanishi R., Kubo N., Ando K., Oki E., Oda Y, & Maehara Y. (2018). CD44v9 is associated with epithelial‐mesenchymal transition and poor outcomes in esophageal squamous cell carcinoma. Cancer Medicine, 7(12), 6258-6268.

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Activation and Culture of Human T Cells

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Cells were cultured in RPMI-1640 medium supplemented with 2 mM glutamine, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, penicillin (50 U ml⁻¹), streptomycin (50 μg ml⁻¹; all from Invitrogen), and 5% (v/v) human serum (Swiss Blood Center, Basel). Human T cells were activated with plate-bound anti-CD3 (5 μg/ml, clone TR66) and anti-CD28 (1 μg/ml, clone CD28.2, BD Biosciences) for 48 h in 96-well Nunc Maxisorb plates. 1.5×10⁵ T cells were plated per well. After 48 h of activation, cells were transferred to 96-well U-bottom plates and cultured in IL-2 containing media (500 U/ml). In experiments, in which T cells that were cultured without a stimulus, 1.5×10⁵ T cells were plated in 96-well U-bottom plates and 200 μ1 of culture medium was added per well. Drugs were added at the following concentrations: CHX (50 μg/ml) and bortezomib/PS341 (10 μM).
For SILAC experiments, SILAC RPMI-1640 (GIBCO) was supplemented with 73 mg/l Lys-8 HCl and 42 mg/l Arg-10 HCl, 2 mM glutamine, 1% (v/v) sodium pyruvate, penicillin (50 U/ml), streptomycin (50 μg/ml; all from Invitrogen), and 5% (v/v) dialyzed human serum.

Wolf T., Jin W., Zoppi G., Vogel I.A., Akhmedov M., Bleck C.K., Beltraminelli T., Rieckmann J.C., Ramirez N.J., Benevento M., Notarbartolo S., Bumann D., Meissner F., Grimbacher B., Mann M., Lanzavecchia A., Sallusto F., Kwee I, & Geiger R. (2020). Dynamics in protein translation sustaining T cell preparedness. Nature immunology, 21(8), 927-937.

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Luminol-Based Monocyte Activation Assay

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Monocytes were resuspended in phenol red free SILAC RPMI 1640 (Gibco, Thermo Fisher) supplemented with 2 mM GlutaMAX (Gibco, Thermo Fisher), 5 mM HEPES (Sigma-Aldrich), 0.133 mM l-arginine (Sigma-Aldrich), 0.27 mM l-lysine (Sigma-Aldrich), and 2.5% FCS in a density of 3 × 10⁵/200 µl and seeded in a white 96-well plate (Nunc, Thermo Fisher). After 2 h of incubation at 37°C and 5% CO₂, luminol reagent (140 µM; Cayman Chemicals) was added, and cells were stimulated with 100 ng/ml LPS. Luminescence from oxidized luminol was measured with a platereader (FluoStar Optima, BMG Labtech) over 180 min. Activation of the AMPK with 1 mM AICAR (Sigma-Aldrich) was done prior to LPS stimulation.

Raulien N., Friedrich K., Strobel S., Rubner S., Baumann S., von Bergen M., Körner A., Krueger M., Rossol M, & Wagner U. (2017). Fatty Acid Oxidation Compensates for Lipopolysaccharide-Induced Warburg Effect in Glucose-Deprived Monocytes. Frontiers in Immunology, 8, 609.

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Activation and Culture of Human T Cells

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