HCT116 VIM RFP

The HCT116 VIM RFP reporter cell line, derived from the parental HCT116 (ATCC CCL-247™) colorectal carcinoma cell line, was created using CRISPR/Cas9 gene editing technology. The HCT116 VIM RFP reporter cell line carries a knock-in red fluorescent protein (RFP) reporter, which was integrated before the stop codon at the last exon of the endogenous vimentin (VIM) gene.

Epithelial to mesenchymal transition (EMT) has been recognized to play an important role in cancer cell metastasis and drug resistance. The EMT pathway is of increasing interest as a novel therapeutic avenue in the treatment of cancer. HCT116 VIM RFP cell line (ATCC CCL-247EMT™) was created using the CRISPR-Cas9 platform, in which a red fluorescent protein (RFP) reporter was integrated before the stop codon at the last exon of the endogenous vimentin gene, a widely used mesenchymal cell marker. The integrity of the VIM RFP knock-in allele has been verified at genomic, transcriptional, and translational levels. In HCT116 VIM RFP cells, vimentin-RFP expression can be robustly turned on in response to miR-200 family inhibitor treatment, as previously reported (Park et al, Genes Dev, 22: 894-907, 2008). In contrast, the expression of E-cadherin, a marker of epithelial cells, is significantly reduced upon miR-200 inhibitor treatment. In addition, hypomethylating agent 5-Aza-2’-Deoxycytidine treatment can induce an increase in VIM RFP expression effectively. The HCT116 VIM RFP cell line could be a useful in vitro cell model for dissecting the molecular switches underlying EMT and for identifying compounds that target EMT in colorectal cancer.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

• 10% Fetal Bovine Serum (FBS; ATCC 30-2020)

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
   Cultures can be established between 2 x 10⁴ and 4 x 10⁴ viable cells/cm².
6. Incubate cultures at 37°C.

The STR profile of the HCT-116 VIM RFP reporter cell line is approximately an 80% match to the parental HCT-116 cell line indicating that the cell lines are related (derived from common ancestry). The STR differences are likely attributed to microsatellite instability in the HT-116 cell line, which is a common feature of this cell line.^1,2,3

1. Ahmed DP, et al. Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis Sep 16;2:e71. doi: 10.1038/oncsis.2013.35, 2013. PubMed: 34042735
2. Gu C, et al. Inositol pyrophosphate profiling of two Hct116 cell lines uncovers variation in Insp8 levels. PLoS One 11(10):e0165286. doi: 10.1371/journal.pone.0165286, 2016. PubMed: 27788189
3. Eshleman JR, et al. Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer. Oncogene 10(1):33-7, 1995. PubMed: 7824277

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