LY6G6D, also known as LY6-D, is a type I transmembrane protein belonging to the immunoglobin (Ig) superfamily, that is comprised of cell-surface proteins involved in the immune system and cellular recognition. And it is known to interact with GRB2 and GRB7 in a phosphorylation-dependent manner thus playing an important role in their downstream signal transduction pathways. Diseases associated with LY6G6D include Glucosephosphate Dehydrogenase Deficiency and Anemia, Nonspherocytic Hemolytic, Due to G6pd Deficiency.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 24 hours
Cell Line Generation
CHOK1 mouse LY6G6D cell line was generated using a lentiviral vector expressing the mouse LY6G6D sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/mL Puromycin) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL.
1. Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage
1. Mallya M, Campbell RD, Aguado B (July 2002). "Transcriptional analysis of a novel cluster of LY-6 family members in the human and mouse major histocompatibility complex: five genes with many splice forms". Genomics. 80 (1): 113–23. doi:10.1006/geno.2002.6794. PMID 12079290.
2. "Entrez Gene: Lymphocyte antigen 6 complex, locus G6E pseudogene)".
3. "Human PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
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