KC-6143

Nalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Line

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Background of Nalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Line

CD79 is a heterodimeric molecule comprising two polypeptide chains, B29 (CD79b) and mb-1 (CD79a). It is physically linked in the surface of B cells to membrane immunoglobulin, forming the B cell antigen receptor complex. Expression of the mb-1 (CD79a) chain has been studied in leukaemias and shown to be present in most B lineage acute lymphoblastic leukaemias (ALL). CD79a and CD79b expression precedes immunoglobulin (Ig) heavy-chain gene rearrangement and CD20 expression during B-cell ontogeny and disappears later than CD20 in the late (plasma cell) stage of B-cell differentiation. Study highlights that CD79A and CD79B are required for surface IgM expression in human B cells. Diseases associated with CD79A/CD79B include Agammaglobulinemia 3, Autosomal Recessive and Agammaglobulinemia 3.
BCR protein with a unique structure having two opposing regulatory activities toward small GTP-binding proteins. The C-terminus is a GTPase-activating protein (GAP) domain which stimulates GTP hydrolysis by RAC1, RAC2 and CDC42. Accelerates the intrinsic rate of GTP hydrolysis of RAC1 or CDC42, leading to down-regulation of the active GTP-bound form. The central Dbl homology (DH) domain functions as guanine nucleotide exchange factor (GEF) that modulates the GTPases CDC42, RHOA and RAC1. Promotes the conversion of CDC42, RHOA and RAC1 from the GDP-bound to the GTP-bound form. The amino terminus contains an intrinsic kinase activity. Functions as an important negative regulator of neuronal RAC1 activity. Regulates macrophage functions such as CSF1-directed motility and phagocytosis through the modulation of RAC1 activity. Plays a major role as a RHOA GEF in keratinocytes being involved in focal adhesion formation and keratinocyte differentiation.

Specifications

Catalog NumberKC-6143
Cell Line NameNalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Line
NCBI/UniProt Accession NumberNM_001783.4, NM_000626.4
Clone Number1#
Host Cell LineNalm6-Luc2
DescriptionStable Nalm6-Luc2 clone expressing exogenous CD79a, CD79b and M22-BCR gene.
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640+20% FBS+10% DMSO
Propagation MediumRPMI1640+10%FBS+1μg/mL Puromycin+7μg/mL Blasticidin S
Selection MarkerPuromycin | Blasticidin S
MorphologyLymphoblast-like (round)
SubcultureSplit saturated culture 1:2~1:3 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative

Cell Line Generation

Nalm6-CD79a-CD79b-M22-BCR-Luc2 cell Line was generated using retrovirus vector expressing CD79a, CD79b and M22-BCR sequence.

Characterization

Figure 1: Characterization of Nalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Line stable clone using FACS.

Figure 2: Characterization of Nalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Lines stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 2: Characterization of Nalm6-CD79a-CD79b-M22-BCR-Luc2 Cell Lines stable clone using PCR.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1 μg/mL Puromycin and 7μg/mL Blasticidin S)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 1:2~1:3 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.

References

1.Astsaturov IA, Matutes E, Morilla R, Seon BK, Mason DY, Farahat N, Catovsky D. Differential expression of B29 (CD79b) and mb-1 (CD79a) proteins in acute lymphoblastic leukaemia. Leukemia. 1996 May;10(5):769-73. PMID: 8656670.
2.Chu PG, Arber DA. CD79: a review. Appl Immunohistochem Mol Morphol. 2001 Jun;9(2):97-106. doi: 10.1097/00129039-200106000-00001. PMID: 11396639.
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