BRAF Mutant-HCC4006

This is a malignant BRAF V600E mutant isogenic line derived from the parental HCC4006 (ATCC CRL-2871) cell line that was isolated from the pleural effusion of a white, adult male patient with non-small cell lung cancer. The c.38G>A knock-in mutation encoding BRAF p.V600E protein expression was generated by utilizing the CRISPR/Cas9 gene editing technology.

The parental cell line HCC4006 has a mutation in the EGFR tyrosine kinase domain (L747 - E749 deletion, A750P) and is sensitive to the EGFR inhibitor, osimertinib. The BRAF V600E mutant isogenic line (ATCC CRL-2871IG-5™) depicts the acquired drug resistance mechanism of BRAF mutation, which was observed in clinical patients who have resistance to osimertinib (the 3rd generation of EGFR inhbitor). This cell line has been validated at the genomic, transcript, and protein bio-functional levels and exhibits significant resistance to the EGFR inhibitor, osimertinib when compared to its parental cell line.

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 150 to 400 x g for 8 to 12 minutes.
4. Carefully aspirate the supernatant and discard, leaving the cell pellet.
5. Gently resuspend the cell pellet with the appropriate amount of complete growth medium and transfer cell suspension into a vented T-75.
6. Place the flask in a 37°C incubator with 5% CO₂ .

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of 0.25% Trypsin/0.53 mM EDTA (ATCC 30-2101) to flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 minutes).
   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 aspirate cells by gently pipetting.
5. Centrifuge the cell suspension at approximately 150 to 400 x g for 8 to 12 minutes to remove dissociation agent.
6. Resuspend the cell pellet in an appropriate amount of complete culture medium.
7. Add appropriate aliquots of the cell suspension to new culture vessels.
   Cultures can be established between 1.0 x 10⁴ and 4.0 x 10⁴ viable cells/cm².
8. Incubate cultures at 37°C.

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