KC-6225

RPMI-8226-SIRPA-KO cell line

61143

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Background of RPMI-8226-SIRPA-KO cell line

The SIRP family of proteins consists of five members; SIRPα, SIRPß1, SIRPß2, SIRPγ and SIRPδ. Two of these, SIRPα (also known as PTPNS1, SHPS1, CD172A and P84) and SIRPγ (also known as CD172b), are known to bind CD47. SIRPα contains an extracellular region with three immunoglobulin superfamily (IgSF) domains, including a NH2-terminal ligand binding V-domain. SIRPα is expressed on all myeloid cell types, including monocytes, macrophages, neutrophils, a subset of dendritic cells and microglia. In addition, SIRPα expression has also been observed in brain tissue, and on a subset of CD8+ T cells during chronic infection.

Specifications

Catalog Number	KC-6225
Cell Line Name	RPMI-8226-SIRPA-KO cell line
Clone Number	2B3
Host Cell Line	RPMI-8226
Description	Stable RPMI-8226-SIRPA-KO cell clone with human SIRPα gene knockout
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS
Selection Marker	NA
Morphology	Lymphoblast
Subculture	Split the saturated culture at a ratio of 1:3-1:4 every 3-4 days; seed out at about 1-3 x 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 32 hours
Mycoplasma Status	Negative

Cell Line Generation

RPMI-8226-SIRPA-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of RPMI-8226-SIRPA-KO cell line stable clone using PCR sequencing.

Figure 2:Characterization of RPMI-8226-SIRPA-KO cell line stable clone using RT-PCR sequencing.

Figure 3:Characterization of RPMI-8226-SIRPA-KO cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS)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 1:3-1:4 every 3-4 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Logtenberg MEW, Scheeren FA, Schumacher TN. The CD47-SIRPα Immune Checkpoint. Immunity. 2020 May 19;52(5):742-752. doi: 10.1016/j.immuni.2020.04.011. PMID: 32433947; PMCID: PMC7340539.