mouse 32d cells  (China Center for Type Culture Collection)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Hematopoietic stem cell gene therapy for the treatment of X-linked agammaglobulinemia

doi: 10.1016/j.omtm.2025.101555

Figure Lengend Snippet: Validation of a Btk site-specific gene editing approach (A) Schematic of Btk editing strategy. The top row shows the Btk gene with exons and introns. In the second row, zooming in on intron 1 and exon 2 of the Btk gene, shows the sites where sgRNAs targeting these regions will allow for Cas9 dsDNA breaks. In the third row, recombinant adeno-associated virus (rAAV) vectors containing a nearly full-length human BTK cDNA with 500-bp homology arms directly flanking the dsDNA break site to allow for homologous recombination at either the intron 1 or exon 2 sites. The BTK cDNA donor also contains a “micro” version of the terminal intron 18, Btk 3′ UTR, and WPRE element. The bottom row depicts the BTK minigene inserted into the endogenous murine Btk locus at the intron 1 or exon 2 sites. (B) Outline displaying 32D and Lin- cell editing timeline. Cells are cultured in either R-10 or SFEM + cytokines for the times listed, then electroporated with Cas9 RNP followed directly by transduction with rAAV6 donor. One week post editing, genomic DNA and total protein lysates are harvested for analysis of allelic disruption, gene integration, and exogenous Btk protein expression. (C and G) Genomic DNA harvested from 32D and Lin- cells, respectively, was PCR amplified with primers flanking the Cas9/sgRNA cut sites and sent for Sanger sequencing analysis of Btk allelic disruption via synthego ICE analysis. The y axis represents the frequency of amplified DNA copies which contained insertions/deletions at the Btk dsDNA break site. n = 3. (D and H) Twenty-four hours post electroporation, 32D and Lin- cells, respectively, were analyzed for viable cell counts using hemacytometers and trypan blue exclusion to determine the acute toxic effects of editing reagents. The y axis represents the average percentage of live cells counted across two individual aliquots of a given sample. (E and I) Genomic DNA harvested from 32D and Lin- cells, respectively, underwent in/out ddPCR analysis using primers and probes that allow for specific quantification of BTK cDNA insertion at its endogenous locus to quantify Btk site-specific integration frequency. The y axis represents the frequency of site-specific Btk integration events normalized to a reference housekeeping gene. n = 3. (F and J) Western blot protein analysis of Btk expression from Btk −/− 32D cells and Btk/Tec −/− Lin- cells, respectively. Cells were lysed using RIPA lysis buffer followed by western blotting and chemiluminescent detection of Btk and Beta-Actin protein expression levels; 32D cells were transfected with equivalent Cas9-RNPs followed by transduction of rAAV6 at MOIs of either 1e5 or 5e5. To determine the role of rAAV6 transduction alone on exogenous Btk expression, Lin- cells either received Cas9-RNP or no electroporation, followed by transduction with rAAV6 at an MOI of 5e5. n = 3.

Article Snippet: 32D cells (ATCC CRL-11346) were electroporated at 85% confluency.

Techniques: Biomarker Discovery, Recombinant, Virus, Homologous Recombination, Cell Culture, Transduction, Disruption, Expressing, Amplification, Sequencing, Electroporation, Western Blot, Lysis, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Potassium Iodide Induces Apoptosis in Salivary Gland Cancer Cells

doi: 10.3390/ijms26115199

Figure Lengend Snippet: KI reduces the viability of SLC5A5-expressing SGC cells. ( A , B ) The fold change in SLC5A5 expression was determined by qPCR in ( A ) human submandibular SG epidermoid carcinoma (A253; n = 5), myeloid hematopoietic (HL-6; n = 2, U937; n = 2), human, and murine endothelial, respectively (HUVEC; n = 2, BMEC1; n = 3), as well as in (HT1080; n = 3) fibrosarcoma cells and ( B ) murine lymphoblast (32D; n = 2) cells, fibroblasts (MS-5; n = 2, 3T3; n = 2), and submandibular SG adenocarcinoma (WR21; n = 3) cells. SLC5A5 gene expression levels were normalized to BETA-ACTIN expression in the same samples, and fold changes were adjusted relative to the expression levels in control samples. ( C , D ) The iodine concentration was assessed in cell lysates of A253 ( C ) and WR21 ( D ) cells 48 h after KI treatment at the indicated concentrations ( n = 4/5 per group). ( E ) Representative light microscopy images of murine WR21 and human A253 SGC cells 48 h after incubation with or without KI (100 μM; scale bar = 100 μm). ( F ) The viability rate of A253 cells treated with the indicated KI concentrations for 48 h was determined by trypan blue exclusion ( n = 8 for the control (co) group and n = 3 for KI 25, 50, 100, and 200 μM groups). ( G , H ) The absolute number of viable and control (co) A253 ( G ) and WR21 cells ( H ) after 48 h in culture, following the addition of KI (100 μM), was determined using the trypan blue exclusion assay ( n = 10 and 5/group for A253 cells and n = 4, 3/group for WR21 cells). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using a one-way ANOVA test (to determine the effects of two independent variables on a dependent variable) or Student’s t -test (to compare the performance of two groups under different conditions), with mean ± SD depicted.

Article Snippet: The basic media for the human submandibular SG epidermoid carcinoma (A253) cells (Cat. HTB-41, ATCC, Manassas, VA, USA) was McCoy’s 5A (Modified) Medium (Cat. 16600082, Gibco, Grand Island, NY, USA); for U-937 human histiocytic lymphoma cells (Cat. CRL-1593.2, ATCC, Manassas, VA, USA), it was RPMI-1640 medium (Cat. 11875093, Gibco, Grand Island, NY, USA); for HL-60 human promyeloblast cells (Cat. CCL-240, ATCC, Manassas, VA, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA); for HUVEC human umbilical venule endothelial cells (Cat. CRL-1730, ATCC, Manassas, VA, USA), it was F-12K Medium (Cat. 21127022, Gibco, Grand Island, NY, USA) supplemented with heparin (Cat. H3393, Sigma, Saint Louis, MO, USA) and ECGS (Cat. CB-40006, Fisher Scientific, Waltham, MA, USA); for BMEC human bone marrow microvascular endothelial cell (Cat. CRL-3421, ATCC, Manassas, VA, USA), it was MCDB-131 medium (Cat. 10372-019, Gibco, Grand Island, NY, USA); for HT-1080 human fibrosarcoma cells (Cat. CCL-121, ATCC, Manassas, VA, USA), it was MEM (Cat. 137-17215, Wako, Osaka, Japan); for WR21 mouse submandibular SG adenocarcinoma cells (Cat. CRL-2189, ATCC, Manassas, VA, USA) and for murine NIH/3T3 fibroblast (Cat. CRL-1658, ATCC, Manassas, VA, USA), it was D-MEM medium (Cat. 044-29765, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); for 32D mouse lymphoblast cells (Cat. CRL-11346, ATCC, Manassas, VA, USA), it was RPMI 1640 (Cat. 11875093, Gibco, Grand Island, NY, USA); and for MS5 murine stromal cells (kindly provided by Dr. MAS Moore, Sloan Kettering Cancer Center, New York, NY, USA), it was IMDM (Cat. 12440053, Gibco, Grand Island, NY, USA).

Techniques: Expressing, Gene Expression, Control, Concentration Assay, Light Microscopy, Incubation, Trypan Blue Exclusion Assay

Journal: Cancer Immunology Research

Article Title: A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy

doi: 10.1158/2326-6066.cir-20-1002

Figure Lengend Snippet: Figure 2. HPFCs induce specific signals downstream of IL-12, IL-15, and IL-18 receptors in reporters and primary human NK cells. A, Dose–response curves of HCW9201 (red) and HCW9207 (purple) showing the concentration of HFPCs required to activate individual cytokine-dependent reporters in 32Db (IL-15–dependent) and HEK-Blue (IL-12p70– or IL-18–dependent) cell lines. Data are from two independent experiments. B, Representative donor NK cell phospho-flow histograms examining the IL-12, IL-15, and IL-18 signaling pathways. Purified NK cells were incubated with 12/15/18, HCW9201, and HCW9207 at 50 or 100 nmol/L for 0.25, 1, or 2 hours, depending upon pathway examined. C, Violin plots of the MFI fold change from baseline. Data are from n ¼ 9 donors analyzed in four independent experiments. Significance was measured by two-way ANOVA. , P ≤0.05; , P ≤0.01; , P ≤0.001. Additional violin plots for pAKT and pERK are shown in Supplementary Fig. S2.

Article Snippet: Daudi cells (ATCC, CCL-213; ref. 18), Raji cells (ATCC, CCL-86; ref. 18), K562 cells (ATCC, CCL-243; CBReGFP; ref. 7), and 32Db cells (ATCC, CRL 11346) transfected with pREP9 (Invitrogen) encoding human IL-15Rb were cultured as described previously (22).

Techniques: Concentration Assay, Protein-Protein interactions, Incubation