For all cell culture experiments, the human colorectal cancer cell line, SW480, was used. It was obtained from the Tissue Culture Facility at University of North Carolina’s Lineberger Comprehensive Cancer Center and is ATCC line CCL228. Cells were maintained in L-15 media (Corning) supplemented with 10% heat-inactivated fetal bovine serum and 1× Pen/Strep (Life Technologies) at 37°C with ambient CO2 levels. For transfection of Drosophila proteins into SW480 cells, Lipofectamine 2000 (Life Technologies) was used for transient transfection following manufacturer’s instructions. All constructs contained the pCMV-backbone and Drosophila genes were inserted using the pCR8/Gateway protocol (Invitrogen) and tagged with GFP, RFP, or Flag as described in Pronobis et al. (2015) (link).
L 15 media
Manufactured by Corning
L-15 media is a cell culture medium designed to support the growth and maintenance of various cell types in vitro. It is a balanced salt solution that provides essential nutrients, vitamins, and growth factors required for cell proliferation and survival. L-15 media is commonly used in applications involving cell biology, tissue engineering, and cellular research.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dmem media, by Corning (1 mentions)
Nahco3, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Sodium selenite, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
Primocin, by InvivoGen (1 mentions)
Horse serum, by Corning (1 mentions)
Putrescine, by Merck Group (1 mentions)
Conalbumin, by Merck Group (1 mentions)
Quikchange, by Agilent Technologies (1 mentions)
4 protocols using l 15 media
1
Transient Transfection of Drosophila Proteins in SW480 Cells
2
Generation and Maintenance of 231MFP and HeLa Cell Lines
3
Astrocyte-Neuron Coculture for Neuroprotection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A confluent monolayer of astrocytes was plated a minimum of 48 hr before viral treatment in two‐chamber slides (Lab Tek, 177380) at a density of 5× 104 cells/cm2. Media was washed off 24 hr after viral induction and motor neurons were seeded on top at a density of 2 × 104 cells/cm2. Cocultures were maintained for additional 72 hr in L15‐complete (L15‐C) (L15 media [Cellgro, 10–045‐CV], 0.63 mg mL−1 NaHCO3 [Fisher, S233500], 5 mg mL−1 insulin [Sigma, I6634], 0.1 mg mL−1 conalbumin [Sigma, C7786], 0.1 mM putrescine [Sigma, P5780], 30 nM sodium selenite [Sigma, S5261], 20 nM progesterone [Sigma, P8783], 20 mM glucose, 0.1 mg mL−1 Primocin (InVivoGen, ant‐pm‐2), and 2% horse serum [Cellgro, 35‐015‐CV].)
4
Axin and APC2 Protein Localization in SW480 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW480 cells were cultured at 37° C at normal atmospheric levels of CO2 in L15-media (Cellgro) supplemented with 10% FBS and 1x penicillin–streptomycin. Drosophila APC2 or Axin protein constructs were transfected into SW480s using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Cells were imaged 24 hours later. Full length Drosophila APC2 and Axin were cloned with either a GFP, RFP, or Flag tag as in [17 (link)].
To verify that Axin:GFP polymerization is not simply a result of di- or oligomerization of the GFP protein, we created a monomeric GFP (mGFP) by changing Alanine 206 to Leucine [46 (link)]. We created the A206K amino acid change in our base plasmid (pCMV-Axin:GFP; [17 (link)]) using QuikChange (Agilent Technologies) following manufacturer’s recommendations. The full plasmid was sequenced to verify the amino acid change in GFP and to ensure no other detrimental mutations were induced in the plasmid.
To verify that Axin:GFP polymerization is not simply a result of di- or oligomerization of the GFP protein, we created a monomeric GFP (mGFP) by changing Alanine 206 to Leucine [46 (link)]. We created the A206K amino acid change in our base plasmid (pCMV-Axin:GFP; [17 (link)]) using QuikChange (Agilent Technologies) following manufacturer’s recommendations. The full plasmid was sequenced to verify the amino acid change in GFP and to ensure no other detrimental mutations were induced in the plasmid.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!