6-well plates were incubated with 1ml poly-D-lysine solution (Sigma, P6407, 10 μg/mL) for 30 minutes at room temperature after which time the poly-lysine solution was removed and the wells washed twice with 2 mL of distilled water. To the wells were added HCT-116 cells, (ATCC, CCL-247), maintained in McCoy’s 5A medium (Invitrogen, 16600-082) supplemented with 10% FBS (Atlanta Biologicals, S11050), and Penicillin-Streptomycin (100 U/mL) (Invitrogen, 15140122), or SW480 cells (ATCC, CCL-228), maintained in MEM growth medium (Sigma, M4655) supplemented with 10% FBS and 100 U per ml penicillin and streptomycin. The cells were allowed to grow for 48 hours at 37°C, 5% CO2 in an incubator. The cell confluency was approximately 60–80%. The media was then replaced with fresh media containing the indicated compounds in DMSO or DMSO. The final DMSO concentration was 0.1%. The cells were incubated for 18 hours. The media was removed and the cells washed 2 times with PBS, and the cytosolic fractions isolated using hypotonic buffer as described previously (Chen M, et. al. Biochemistry (2009) 48(43): 10267–10274). Cytosolic fractions were analyzed by Western blot using antibodies to β-catenin (Santa Cruz Biotechnology, SC-7963) or Axin2 (Santa Cruz Biotechnology, SC-20784). As a loading control, β-actin was analyzed with antibodies to β-actin (C-4, Santa Cruz Biotechnology, SC-47778).
Mem growth medium
Manufactured by Merck Group
Sourced in United States
MEM growth medium is a cell culture medium developed by Merck Group. It is formulated to support the growth and maintenance of a variety of mammalian cell lines in vitro. The medium provides the necessary nutrients, vitamins, and other components required for cell proliferation and survival.
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Lab products found in correlation
2 protocols using mem growth medium
1
Cytosolic β-catenin and Axin2 Analysis
2
Cell Invasion Assay for Bacterial Isolates
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Bacterial isolates were thawed from frozen stocks of the strain library of the Research Unit on Implant Infections and plated on Tryptic Soy Agar (TSA, Biolife). For the cell invasion assays, bacteria were grown in Tryptose Broth (TB) (Biolife) at 37 °C for 18 h. Bacterial cultures were centrifuged at 4000 RCF for 15 min and the pellets obtained were resuspended in MEM growth medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 2 mM L-glutamine (Sigma-Aldrich). Bacterial concentration was estimated by optical density (OD) at 550 nm using a Hewlett Packard G1103A spectrophotometer. However, for the accurate assessment of the titre of viable bacteria, all starting bacterial suspensions were quantified in terms of CFU by agar plating. Only this a posteriori enumeration of CFU of the inoculum was used for studying the relationship between multiplicity of infection (MOI) and internalised CFU. Up to 17 serial 1:2 dilutions (last corresponding to a dilution factor of 1:65,536) were prepared starting from the initial suspension. For each single bacterial strain, up to 5 independent bacterial assays were performed and plotted together.
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