human jurkat t cells  (ATCC)

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: Timed delivery of fluorescently tagged dextran to Jurkat cells (A) Schematic of typical and timed delivery experiments performed. In a typical experiment, target cells are premixed with fluorescent cargo (fluorescein isothiocyanate dextran, FITC-Dex). In timed delivery experiments, cells are treated in delivery buffer without cargo while FITC-Dex is added at a later time point. Flow cytometry is performed within 2 h of treatment to determine delivery efficiency (percentage of cells positive for FITC). (B) Filtroporation of Jurkat cells with FITC-Dex; cargo was either premixed or added at the specified time point. Controls were untreated, mock filtroporated (FP without cargo) or incubated with FITC-Dex without FP (Incub Ctrl). (C) Viabilities determined by 4′,6-diamidino-2-phenylindole (DAPI) staining at the time of flow cytometry after FP experiments. (D) Nucleofection (EP) of Jurkat cells with FITC-Dex; cargo was either premixed or added at the specified time point. Controls were untreated or mock nucleofected (EP without cargo). (E) Viabilities determined by 4′,6-diamidino-2-phenylindole (DAPI) staining at the time of flow cytometry after EP experiments. Data are shown for n ≥ 2 independent experiments. See also and . Data are shown as mean ± standard deviation.

Article Snippet: Human: Jurkat Clone E6-1 , ATCC , TIB-152.

Techniques: Flow Cytometry, Incubation, Staining, Standard Deviation

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: Calcium concentration is critical during transfection by filtroporation (A) Delivery efficiency determined by flow cytometry within 2 h of filtroporation of Jurkat or CD34 + bone marrow derived human hematopoietic stem and progenitor cells (HSPCs) with fluorescein isothiocyanate (FITC)-tagged dextran (FITC-Dex) in cell media with low calcium (Roswell Park Memorial Institute, RPMI), FP-Dex (0.42 mM Ca 2+ ), or with additional 1 mM CaCl 2 added, FP-Dex (1.42 mM Ca 2+ ). Controls were either untreated, filtroporated without cargo (FP-Mock), or incubated in FITC-Dex without FP (Incub-Dex). (B) Viabilities of filtroporated Jurkat cells determined by 4′,6-diamidino-2-phenylindole (DAPI) at the time of flow cytometry. Cells were also transfected in phosphate buffered saline (PBS) as delivery buffer (FP-Dex-PBS). (C) Jurkat cells were filtroporated with green fluorescent protein (GFP)-encoding plasmid (pGFP) in different delivery buffers: RPMI, minimum essential media (MEM) containing no calcium, or a 1:1 mixture of RPMI and MEM (0.21 mM Ca 2+ ). Media was introduced at the bottom of the collection tube such that cells subjected to filtroporation fell into either RPMI, MEM or RPMI:MEM 1:1 media. Results are displayed for cells at 24 h post-filtroporation (timepoint with highest expression). (D) Cell counts at 24–72 h post-filtroporation performed with acridine orange/propidium iodide fluorescent staining. (E) Percentage of GFP-positive cells (left) and cell viability (right) determined by DAPI staining at the time of flow cytometry. Mock RPMI/MEM: cells filtroporated without cargo in RPMI or MEM; FP-RPMI-pGFP: cells subjected to FP in RPMI with pGFP cargo; FP-MEM-pGFP: cells subjected to FP in MEM with pGFP cargo; FP-MtR-pGFP: cells subjected to FP with pGFP cargo in MEM falling into RPMI media; FP-Premix-pGFP: cells subjected to FP with pGFP cargo in RPMI:MEM premixed media. Data are shown for n ≥ 2 independent experiments. (∗∗ p < 0.005, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001). Data are shown as mean ± standard deviation.

Article Snippet: Human: Jurkat Clone E6-1 , ATCC , TIB-152.

Techniques: Concentration Assay, Transfection, Flow Cytometry, Derivative Assay, Incubation, Saline, Plasmid Preparation, Expressing, Staining, Standard Deviation

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: SNAP23 and CHMP4B are involved in membrane repair immediately after filtroporation (A–H) Confocal images overlaying enhanced green fluorescent protein (eGFP) and 4′,6-diamino-2-phenylindole (DAPI) fluorescence channels for untreated and filtroporated Jurkat cells expressing (A,E) GRAF1-eGFP, (B,F) SNAP23-eGFP, and (C,G) CHMP4B-mCherry, with (D,H) free cytoplasmic eGFP as a negative control. (I–P) Quantification of foci per cell per image captured on confocal microscopy for the different cell lines with cells immediately fixed after filtroporation or at different time points (GRAF1-eGFP: I and M; SNAP23-eGFP: J and N; CHMP4B-mCherry: K and O; free cytoplasmic eGFP: L and P). (∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). See also . Data are shown as mean ± standard deviation. Scale bars are 10 μm in panels A and E, and 5 μm in all other images.

Article Snippet: Human: Jurkat Clone E6-1 , ATCC , TIB-152.

Techniques: Membrane, Fluorescence, Expressing, Negative Control, Confocal Microscopy, Standard Deviation