cell lines k562 cells american type culture collection n a kbm5 cells jin  (ATCC)

Journal: The Journal of Immunology Author Choice

Article Title: The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk

doi: 10.4049/jimmunol.2300406

Figure Lengend Snippet: TRPML1 deficiency impairs autophagy, lysosomal arming, and migration of NK cells. ( A ) Relative AMPK phosphorylation of primary NK cells following 5-min stimulation with the TRPML1 agonist MK6-83 (20 μM). Data are shown as the p-AMPK (mean fluorescence intensity [MFI])/AMPK (MFI) ratio. Pooled analysis of is from n = 3 donors. ( B ) Autophagy induction measured by flow cytometry–based evaluation of membrane-bound LC3 in NK92 cells following treatment with the AMPK agonist A-769662 (300 μM). Data represent three independent biological replicates. ( C ) Representative immunoblot of the autophagy markers LC3-I and LC3-II in NK92 WT cells. Actin was used as a loading control. ( D ) Immunoblot quantification of LC3 levels (fold change) following stimulation with DMSO or MK6-83 (10 μM) for 3 h in NK92 cells. Three independent experiments were analyzed. ( E ) Autophagy induction measured by flow cytometry–based evaluation of membrane-bound LC3 in NK92 cells following treatment with the TRPML1 agonist MK6-83. Data represent three independent biological replicates. ( F ) Comparative analysis of baseline levels of autophagic vacuoles, as determined by flow cytometry of CYTO-ID autophagy detection kit–stained primary NK cells transfected with mock small interfering RNA (siRNA) as control or siRNA targeting MCOLN1 . Data are shown from six donors. ( G ) Transwell migration assay of NK92 cells. Cells were either left unstimulated or migrated toward a gradient of the chemokine CXCL12 (100 ng/ml) for 1 h. Each dot represents an independent experiment. Data are presented as mean ± SEM of quadruplicates. ( H ) Intracellular cytokine responses of primary NK cells challenged with K562 cells (6-h coculture) in the presence of a TRPML1 antagonist (ML-SI3) in the indicated concentrations. CCCP was used to compromise mitochondrial function. Data are shown from six donors. Paired t tests were performed in graphs (A–G) and a one-way ANOVA followed by a Dunnett post hoc test was used for (H). * p < 0.05, ** p < 0.01, **** p < 0.0001. ns, not significant.

Article Snippet: For the cytotoxicity assay, K562 cells (American Type Culture Collection) cells transfected with IncuCyte NucLight Green lentivirus reagent (Sartorius) for stable expression of nuclear GFP were seeded at a density of 5 × 10 6 cells/well in a coated 96-well plate.

Techniques: Migration, Phospho-proteomics, Fluorescence, Flow Cytometry, Membrane, Western Blot, Control, Staining, Transfection, Small Interfering RNA, Transwell Migration Assay

Journal: Blood Cancer Journal

Article Title: Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice

doi: 10.1038/bcj.2016.38

Figure Lengend Snippet: Blinatumomab BiTEs were secreted from RNA-transferred T cells and bound to T cells for tumor recognition. The T cells were electroporated with an RNA encoding CAR19 (CAR RNA), blinatumomab BiTEs (Blina-RNA) or GFP (at an RNA dose of 10 μg of RNA per 0.1 ml of T cells per electroporation). Eighteen hours after electroporation, the T cells were stained with a goat anti-mouse IgG Fab (mIgG Fab) to detect the expression of the CAR or blinatumomab on the T-cell surface (gated on CD3 + T cells) ( a ). Eighteen hours after electroporation, the CAR RNA or BiTE RNA T cells alone or mixed with an equal amount of GFP RNA T cells (GFP) were tested for their lytic activity using a cytotoxic T-lymphocyte assay at the effector:target ratio of 5:1 ( b ). The supernatant from the Blina-RNA T cells (Blina-RNA Sup.) was collected 18 h after electroporation, diluted 10 (1/10) or 100 times (1/100) with culture medium, added to T cells that were not electroporated with any RNA (No RNA) and co-cultured with the CD19 + cell lines (Nalm6, K562-CD19 or Raji cells). The K562 cell line was used as a negative control. T cells that had been electroporated with the CAR RNA or Blina-RNA were used as positive controls in the CD107a assay (gated on CD8 + T cells) ( c ) (representative of three independent experiments). * P <0.05; ** P <0.01.

Article Snippet: The Nalm6 (DSMZ, Braunschweig, Germany), Raji (American Type Culture Collection, Manassas, VA, USA) and K562 (American Type Culture Collection) cell lines were cultured per the providers' instructions.

Techniques: Electroporation, Staining, Expressing, Activity Assay, Cytotoxic T Lymphocyte Assay, Cell Culture, Negative Control

Journal: Blood Cancer Journal

Article Title: Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice

doi: 10.1038/bcj.2016.38

Figure Lengend Snippet: Blinatumomab BiTE RNA T cells were less dependent on co-stimulation and exhibited enhanced division and proliferation. CFSE-labeled resting CD4 T cells were electroporated with the blinatumomab BiTE RNA (Blina-RNA) or CD19-BBZ CAR RNA (CAR RNA) at the indicated RNA doses and stimulated with either irradiated K562-CD19 cells mixed with an equal amount of irradiated K562 cells (as control for K562-CD86; K-19/K562) or irradiated K562-CD19 cells mixed with an equal amount of irradiated K562-CD86 cells (K-19/K-86). The CFSE dilution was examined at day 6 (gated on CD3 + T cells). The results from a representative experiment are shown in a , and a summary of three independent experiments is shown in b . CFSE-labeled CD45RO + (memory) or CD45RO − (naive) resting CD4 T cells were electroporated with the blinatumomab BiTE RNA (Blina-RNA) or CD19-BBZ RNA (CAR RNA) at the indicated RNA doses and stimulated with either irradiated K562-CD19 cells mixed with an equal amount of irradiated K562 cells (as control for K562-CD86) or irradiated K562-CD19 cells mixed with an equal amount of irradiated K562-CD86 cells. The CFSE dilution was examined at day 6 (gated on CD3 + T cells) ( c ), and the T-cell expansion was monitored at different days after stimulation ( d ). The CSFE-labeled T cells that had been electroporated with either 1 or 5 μg of the CD19-BBZ (19BBZ), CD19-28Z (19-28Z) or blinatumomab BiTE (Blina-RNA) RNA were stimulated with irradiated K562, K562-CD19/K562 or K562-CD19/K562-CD86 cells. Six days later, the T cells were subjected to the flow cytometry analysis for CFSE dilution (gated on CD3 + T cells) ( e ). Five hundred thousand T cells that had been electroporated with 5 μg of the CD19-BBZ (19BBZ), CD19-28Z (19-28Z) or blinatumomab BiTE (Blina-RNA) RNA were stimulated with K562-CD19/K562 or K562-CD19/K562-CD86 cells. Six days later, the total number of viable T cells was counted, and the fold increase in T-cell expansion was calculated ( f ) (representative of two independent experiments). NS, not significant. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The Nalm6 (DSMZ, Braunschweig, Germany), Raji (American Type Culture Collection, Manassas, VA, USA) and K562 (American Type Culture Collection) cell lines were cultured per the providers' instructions.

Techniques: Labeling, Irradiation, Control, Flow Cytometry