KC-3782

BaF3-MPL-CALR-del52 Cell Line

Stable Ba/F3 cell line expressing exogenous MPL & CALR-del52 gene

Home » BaF3-MPL-CALR-del52 Cell Line

Background

MPL, also known as TPO-R, is expressed predominantly on the surface of MKs, platelets, hemangioblasts, and hematopoietic stem cells (HSCs). Binding of TPO to the megakaryocyte TPO-R leads to different effects: prevention of megakaryocyte apoptosis; increased megakaryocyte number, size, and ploidy; increasing rate of megakaryocyte maturation; and internalization of the TPO/TPO-R complex. Thrombopoietin R involved in multiple signal transduction pathways, such as JAK, STAT, and MAP kinase. Calreticulin is a multifunctional soluble protein that binds Ca2+ ions (a second messenger in signal transduction), rendering it inactive. The Ca2+ is bound with low affinity, but high capacity, and can be released on a signal. Calreticulin is located in storage compartments associated with the endoplasmic reticulum and is considered an ER resident protein. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number:
KC-3782
Cell Line Name:
BaF3-MPL-CALR-del52 Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Description:
Stable Ba/F3 cell line expressing exogenous MPL & CALR-del52 gene
Quantity:
One vial of frozen cells (5X106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium:
RPMI1640 + 10%FBS
Selection Marker:
Puromycin
Morphology:
Mostly single, round (some polymorph) cells in suspension
Subculture:
Split the saturated culture at a ratio of 1:4~1:8 every 2~3 days; seed out at about 1-2 x 105 cells/ml
Incubation:
37 °C with 5% CO2
Storage:
Liquid nitrogen immediately upon receiving
Doubling Time:
Approximately 20 hours
Mycoplasma Status:
Negative

Cell Line Generation

BaF3-MPL-CALR-del52 Cell Line was generated using a lentiviral vector expressing the MPL and CALR-del52 sequence.

Characterization

Figure: Characterization of BaF3-MPL-CALR-del52 Cell Line stable clone using PCR sequencing.

Application

Hybridoma or ligand binding screening with FACS.

Cell Resuscitation

1. Prewarm the culture medium (RPMI164 supplemented with 10% FBS) 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 to a 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 9.0 ml complete culture medium. 5. Spin at ~ 125 x g for 5~7 minutes at room temperature and discard the supernatant without disturbing the pellet. 6. Resuspend the cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask. 7. Incubate the flask in an incubator at 37°C, 5% CO2. 8. Split the saturated culture at a ratio of 1:4 ~ 1:8 every 2~3 days; seed out at about 1-2 x 105 cells/ml.

Cell Freezing

1. Freshly prepare the freezing medium (70% RPMI164 + 20% FBS + 10% DMSO) before use. 2. Keep the freezing medium on ice and label cryovials for later use. 3. Trypsinize and harvest cells to a sterile, conical centrifuge tube during the logarithmic growth period and count the cells. 4. Centrifuge the cells at room temperature at 250 x g for 5 minutes and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3 x106 cells/ml in the chilled freezing medium. 6. Aliquot 1ml 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.

References

1.       McCauliffe DP, Zappi E, Lieu TS, Michalak M, Sontheimer RD, Capra JD (Jul 1990). "A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia "memory molecule"". The Journal of Clinical Investigation. 86 (1): 332–5. doi:10.1172/JCI114704. PMC 296725. PMID 2365822. 2.       "Entrez Gene: calreticulin".
Please enable JavaScript in your browser to complete this form.