Nalm6-Fluc-Puro (Imanis #CL151) cells were transfected with Cas9-gRNA ribonucleoprotein complexes targeting human CD19. Individual cell clones were generated using a methylcellulose based semi-solid medium and screened for CD19 expression by flow cytometry. Sequencing of the targeted CD19 region confirmed genetic knock-out in the selected cell clone. 

This cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

The parental Nalm6 cell line was licensed and purchased directly from ATCC*.

Replication Competent Lentivirus (RCL) Test (including a test report) is available for this cell line at an added cost. Contact us to learn more.

This cell line is also available for purchase as a pair with the Nalm6-Fluc-Puro (CD19 positive) cell line, for a discounted rate (link). 

*The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection.

Morphology

Cell photos taken at 200x magnification

Luciferase Activity

The indicated number of cells were placed in wells of a 96-well plate. After the addition of 3 mg/mL d-luciferin, the plate was immediately imaged using a microplate reader. The total relative light units (RLU) was plotted as a function of cell number.

CD19 Expression

Nalm6-Fluc-Puro/CD19-KO cells were stained with isotype control antibody (grey) or antibody specific to CD19 (green) and analyzed by flow cytometry.

Surface Receptor Profiling

Nalm6-Fluc-Puro/CD19-KO (green) cells were stained with isotype control antibody (grey) or antibody specific to BCMA (A), CD20 (B), GPRC5D (C), or CD38 (D) and analyzed by flow cytometry.

CD19-CAR Activation Assay

CD3+ Jurkat cells expressing a CD19-CAR and GFP (Jurkat-CD19-CAR-GFP) or GFP alone (Jurkat-GFP) were co-cultured with either Nalm6-Fluc-Puro (Imanis #CL151) or Nalm6-Fluc-Puro/CD19-KO (Imanis #CL180) target cells. After 48 hours, cells were analyzed by flow cytometry; CD3+ cells were gated for GFP and CD69, a marker of T-cell activation.

Complete Growth Medium: RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 1% Penicillin/Streptomycin.

For long-term maintenance (more than two to three weeks) addition of 1 µg/mL of puromycin to the media is recommended to maintain high luciferase expression.

These cells should be grown in the indicated medium and subcultured as needed to maintain a density between 3 x 10⁵ and 3 x 10⁶ cells/mL. The cells can be passaged by dilution in fresh medium, with occasional passaging using centrifugation to limit the amount of debris in cultures.

These cells are suitable for in vitro and in vivo experimentation. The Fluc transgene facilitates in vivo noninvasive bioluminescent imaging of implanted cells and quantitation of cells in vitro. 

These cells were transduced with a self-inactivating (SIN) lentiviral vector in which the viral enhancer and promoter have been deleted. Transcription inactivation of the LTR in the SIN provirus increases biosafety by preventing mobilization by replication competent viruses and enables regulated expression of the genes from the internal promoters without cis-acting effects of the LTR1. Nevertheless, all work with these cells should be performed under biosafety-level 2 (BSL2) conditions by trained personnel. Institutional requirements may permit handling of these cells under BSL1 conditions if certain criteria are met.

1. Miyoshi, H et al. (1998). Development of a self-inactivating lentivirus vector. Journal of Virology 72: 8150-8157.