154cf media

Manufactured by Thermo Fisher Scientific

154CF media is a general purpose culture medium designed for the growth of a wide range of microorganisms. It provides the necessary nutrients and growth factors to support the cultivation of bacteria, yeasts, and other microbes in a laboratory setting. The composition and formulation of 154CF media is intended to promote the optimal growth and reproduction of a diverse range of microbial species.

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Lab products found in correlation

In fusion cloning kit, by Takara Bio (1 mentions) Pegfp c1, by Takara Bio (1 mentions) Pegfp c1, by Thermo Fisher Scientific (1 mentions) Fugene 6 transfection reagent, by Promega (1 mentions) Mef 1 nucleofector kit, by Lonza (1 mentions) Smoothened agonist sag, by Merck Group (1 mentions) Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)

4 protocols using 154cf media

Cell Culture Conditions for BCC and Other Cells

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ASZ001 and BSZ001 BCC cells were cultured in 154CF media (Life
Technologies) supplemented with 2% chelated fetal bovine serum and
0.05mM CaCl₂. Experiments carried out using low serum conditions
contained 154CF media containing 0.2% chelated FBS and 0.5nM
CaCl₂. NIH-3T3, HaCaT, and HEK-293T cells were cultured in DMEM
supplemented with 10% fetal bovine serum. Hedgehog induction experiments
carried out using low-serum DMEM containing 0.5% FBS. UW-BCC1 cells
(human BCC cells) were isolated from a patient with superficial BCC. Cells were
cultured as described previously^{55 (link)}.

Whitson R.J., Lee A., Urman N.M., Mirza A., Yao C.Y., Brown A.S., Li J.R., Shankar G., Fry M.A., Atwood S.X., Hollmig S.T., Aasi S.Z., Sarin K.Y., Epstein EH J.r., Tang J.Y, & Oro A.E. (2018). Non-canonical hedgehog pathway activation by MKL1/SRF promotes drug-resistance in basal cell carcinomas. Nature medicine, 24(3), 271-281.

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Generation and Characterization of Sufu Variants

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Sequence verified SUFU variants were created using the InFusion cloning kit (Clontech) and cloned into peGFP-C1 (Clontech) and pCDH (SBI) vectors. Sufu-null MEFs [2 (link)] and HEK-293T cells were cultured in DMEM media supplemented with 10% fetal bovine serum. ASZ001 [9 (link)] were cultured in 154CF media (Life Technologies) supplemented with 2% chelated fetal bovine serum and 0.05mM CaCl₂.
For qRT-PCR experiments, each peGFP-C1-SUFU construct was nucleofected into Sufu-null MEFs using the Amaxa nucleofection kit and protocol, grown to confluency in DMEM + 10% fetal bovine serum, and then withdrawn from serum in DMEM for 24 hours to ensure each cell was in the G0 phase of the cell cycle. Nucleofection was done with 1 μg of DNA into 100 uL of media containing Sufu-null MEF cells and nucleofection reagent. After nucleofection, cells were plated onto a 24-well plate with 1 mL of DMEM supplemented with 10% fetal bovine serum per well. Cells were incubated for 24 hours then RNA was purified for qRT-PCR.

Urman N.M., Mirza A., Atwood S.X., Whitson R.J., Sarin K.Y., Tang J.Y, & Oro A.E. (2016). Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions. PLoS ONE, 11(12), e0168031.

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Hedgehog Signaling Assays in Cell Lines

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ASZ001 (ASZ, gender: female) BCC cells were cultured in 154CF media (Life Technologies) supplemented with 2% chelated fetal bovine serum, Human Kerytinocyte Growth Supplement (Thermo Fisher), Penn-strep, and 0.05mM CaCl2. Experiments assaying hedgehog signaling carried out in serum-free conditions.
NIH-3T3 (gender: male) and HEK-293T (gender: female) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Experiments assaying hedgehog signaling were carried out in 0–0.2% FBS supplemented with Smoothened Agonist (SAG, Sigma).
Mammalian cell transfection performed using FuGENE® 6 Transfection Reagent (Promega), Lipofectamine® LTX with Plus™ Reagent (Thermo Fisher), and MEF 1 Nucleofector® Kit (Lonza) per manufacturer protocol. Transient transfection mammalian expression vectors are included below. Stable expression produced by piggybac transposition.

Mirza A.N., McKellar S.A., Urman N.M., Brown A.S., Hollmig T., Aasi S.Z, & Oro A.E. (2018). LAP2 Proteins Chaperone GLI1 Movement Between Lamina and Chromatin to Regulate Transcription. Cell, 176(1-2), 198-212.e15.

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Cell Culture Conditions for BCC and Other Cells

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