KC-0227

293T-HVEM-Cell-Line

20053

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Background of 293T-HVEM-Cell-Line

HVEM is also known as TNFRSF14, is a type I membrane protein belonging to the TNF/NGF receptor superfamily. HVEM expression has been detected in peripheral blood T cells, B cells, monocytes and in various tissues enriched in lymphoid cells. The extracellular domain of HVEM has been shown to interact directly with the herpes simplex virus envelope glycoprotein D (gD).

Specifications

Catalog Number	KC-0227
Cell Line Name	293T-HVEM-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T cell line expressing exogenous human HVEM gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+0.5µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T human HVEM cell line was generated using a lentiviral vector expressing the human HVEM sequence.

Characterization

Figure: Characterization of HVEM overexpression in 293T stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 0.5μg/mL puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Kwon BS, Tan KB, Ni J, Oh KO, Lee ZH, Kim KK, Kim YJ, Wang S, Gentz R, Yu GL, Harrop J, Lyn SD, Silverman C, Porter TG, Truneh A, Young PR (May 1997). "A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation". The Journal of Biological Chemistry. 272 (22): 14272ÿ6.
"Entrez Gene: TNFRSF14 tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator)".
Ware, Carl (2008). "Chapter 25: TNF-Related Cytokines in Immunity". In Paul, William (ed.). Fundamental Immunology (Book) (6th ed.). Philadelphia: Lippincott Williams & Wilkins. pp. 776ÿ801. ISBN 978-0-7817- 6519-0.
Hoek KS, Schlegel NC, Eichhoff OM, Widmer DS, Praetorius C, Einarsson SO, Valgeirsdottir S, Bergsteinsdottir K, Schepsky A, Dummer R, Steingrimsson E (December 2008). "Novel MITF targets identified using a two-step DNA microarray strategy". Pigment Cell & Melanoma Research. 21 (6): 665ÿ76.