KC-0992

293T-CD33-Cell-Line

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Background of 293T-CD33-Cell-Line

CD33, also known as siglec-3, is transmembrane protein belonging to the SIGLEC family of lectins, which is mainly expressed on the surface of myeloid cells. CD33 can inhibits tyrosine phosphorylation and calcium mobilization, suggesting Siglec-3 can mediate inhibitory signals in immune response. CD33 is also the target of several antibodies in tumor therapy, including gemtuzumab ozogamicin approved in 2017, and vadastuximab talirine.

Specifications

Catalog Number	KC-0992
Cell Line Name	293T-CD33-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CD33 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 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-CD33-cell-line was generated using a lentiviral vector expressing the CD33 sequence.

Characterization

Figure 1: Characterization of CD33 overexpression in the 293T-CD33 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:8 every 2-3 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

Garnache-Ottou, Francine, Laurence Chaperot, Sabeha Biichle, Christophe Ferrand, Jean-Paul Remy-Martin, Eric Deconinck, Patrick Darodes de Tailly, et al. 2005. “Expression of the Myeloid-Associated Marker CD33 Is Not an Exclusive Factor for Leukemic Plasmacytoid Dendritic Cells.” Blood 105 (3). American Society of Hematology: 1256–64. doi:10.1182/blood-2004-06-2416.
Hernández-Caselles, Trinidad, María Martínez-Esparza, Ana B Pérez-Oliva, Ana M Quintanilla-Cecconi, Ana García-Alonso, D María Rocío Alvarez-López, and Pilar García-Peñarrubia. 2006. “A Study of CD33 (SIGLEC-3) Antigen Expression and Function on Activated Human T and NK Cells: Two Isoforms of CD33 Are Generated by Alternative Splicing.” Journal of Leukocyte Biology 79 (1). Wiley-Blackwell: 46–58. doi:10.1189/jlb.0205096.
Walter, Roland B, Ted A Gooley, Vincent H J van der Velden, Michael R Loken, Jacques J M van Dongen, David A Flowers, Irwin D Bernstein, and Frederick R Appelbaum. 2007. “CD33 Expression and P-Glycoprotein- Mediated Drug Efflux Inversely Correlate and Predict Clinical Outcome in Patients with Acute Myeloid Leukemia Treated with Gemtuzumab Ozogamicin Monotherapy.” Blood 109 (10). American Society of
Hematology: 4168–70. doi:10.1182/blood-2006-09-047399.