p3 primary cell nucleofection solution  (Lonza)

Journal: Nature Communications

Article Title: PTPN23-dependent ESCRT machinery functions as a cell death checkpoint

doi: 10.1038/s41467-024-54749-2

Figure Lengend Snippet: a Workflow for crRNA-Cas9-RNP-mediated knockout of Ptpn23 in mouse BMDM cells. The CRISPR RNAs (crRNAs) targeting Ptpn23 were consistent with those employed in the mouse AML validation study. Created with Adobe Illustrator and BioRender.com (License reference: https://BioRender.com/p57w564 ). b Immunoblotting analysis of NF-κB and MAPK pathway in BMDM cells harvested 6 days post-nucleofection with crRosa, crN23-1, or crN23-2 ( n = 3 mice). c CTG luminescence cell viability assay in WT or Ptpn23 knockout BMDM cells ( n = 5 mice). Statistical analysis was conducted via two-way ANOVA followed by Tukey’s multiple comparisons test. d Left, Cell death measured by Sytox Green staining in cells cultured for 9 days post-nucleofection ( n = 40 fields from 4 independent experiments). Scale bar: 40 μm. Right, Quantification of Sytox Green positivity. Data are mean ± SEM, analyzed using one-way ANOVA followed by Tukey’s test. e Immunoblot of apoptosis markers in BMDMs treated with 50 ng/ml TNF-α for 4 h, 6-7 days post-nucleofection ( n = 3 mice). f CTG assay in WT and Ptpn23 -knockout BMDMs pretreated with 40 μM z-VAD-fmk for 1 h, followed by treatment with 1 μg/ml TNF-α for the indicated time, 6 days post-nucleofection. g Immunoblot of MLKL in BMDMs 7 days post-nucleofection with crRosa or crN23, treated with 40 μM z-VAD-fmk for 1 h, followed by 1 μg/ml TNF-α for 4 h ( n = 2 independent experiments, each using cells from three mice). Immunoblots in ( b , e , and g ) showed consistent results across three, three, and two independent experiments, respectively, with representative blots presented. h CTG assay in WT and Ptpn23 -knockout BMDMs primed with 100 ng/ml LPS for 4 h, followed by 2.5 μM nigericin for 4 h, 6 days post-nucleofection. Data for ( f , h ) are mean ± SEM from six replicates derived from three mice, analyzed using two-way ANOVA followed by Tukey’s multiple comparisons tests. i Proposed model: PTPN23 collaborates with NAP1 to facilitate receptor sorting. On the right-hand side, representing the normal condition, this process involves the engagement of ESCRT, leading to the lysosomal degradation of death receptors. On the left-hand side, loss of PTPN23 results in the prolonged accumulation of death receptors within the endosomal compartment, triggering the activation of multiple cell death pathways. Created with Adobe Illustrator and BioRender.com (License reference: https://BioRender.com/x06g968 ).

Article Snippet: Following a PBS wash and cell counting, 10 6 cells per reaction were resuspended in 20 μl of P3 primary nucleofection solution (V4XP-3032, P3 Primary Cell 4D-NucleofectorTM X Kit S, Lonza).

Techniques: Knock-Out, CRISPR, Biomarker Discovery, Western Blot, Viability Assay, Staining, Cell Culture, CTG Assay, Derivative Assay, Activation Assay