Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: A) Freshly isolated CD8+ T cells were pretreated with Kv channel blockers, ShK (10 nM), MgTx (30 nM) and ChTx (50 nM), for 3 hours, then stimulated with anti-CD3 alone or anti-CD3/CD28. After 4 days of culture, proliferation was measured by [3H] thymidine uptake. Data show the mean ± SD of three experiments. Significant differences are marked as follows: (*, p <0.05; **, p <0.01; ***, p <0.005). (B). Isolated CD8+ T cells were labeled with PKH26 stimulated with anti-CD3/CD28 for 24 h, and then transduced with a lentiviral vector encoding the dominant-negative Kv1.x or the GFP control alone. PKH26 fluorescence was analyzed by flow cytometry at baseline and 5 and 11 days later as shown. Quantification of proliferating cells was evaluated by gating on PKH26high PKH26dim and PKH26low among GFP+ cells. (C). Transduced CD8+ T cells were stained with anti-CD8, anti-CCR7 or anti-CD45RA mAbs seven days after transduction and analyzed for the percentages of naïve, T CM , T EMRA and T EM cells in gated GFP+ CD8+ cells. FACS plots shown are representative data from three separate experiments using cells from different donors. (D) The percentages of each CD8+ subset displaying GFP fluorescence are presented as mean ± SD of three experiments. Values that are significantly different from that of GFP control are indicated as follows: **, p <0.01; ***, p <0.005.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Labeling, Transduction, Plasmid Preparation, Dominant Negative Mutation, Fluorescence, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: Freshly isolated CD8+ T cells were pretreated with a Kv1.3 channel blocker, ShK at various concentrations (A) or with ShK (10 nM), MgTX (30 nM), ChTX (50 nM) and TRAM-34 (500 nM) (B) for 3 h, followed by stimulation with anti-CD3/CD28 or anti-CD3 alone. The levels of GrB were measured in cell supernatants by ELISA at 6 h (A) and indicated times (B and C). Data are mean of triplicate ± SD of one representative of three independent and reproducible experiments. Values that are significantly different from that of non-blocker vehicle treated control are indicated as follows: *, p <0.05; **, p <0.01.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: (A) Freshly isolated CD8+ T cells were stimulated with anti-CD3/CD28 or anti-CD3 for 24 hours. Cells were then stained with a CD107a-specific mAb, or an IgG1 isotype control (filled histogram) at the indicated times. (B). CD8+ T cells were pretreated with ShK (10 nM), MgTX (30 nM), ChTX (50 nM) and TRAM-34 (500 nM) for 3 h, followed by stimulation with anti-CD3/CD28 or anti-CD3 alone for 6 hours. Surface expression levels of CD107a were then analyzed by flow cytometry. FACS plots shown are representative data from three separate experiments.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Staining, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: Freshly isolated human CD8+ T cells were simulated with anti-CD3 or anti-CD3/CD28 in the presence or absence of MgTx. Cultured supernatants were collected at 3 days after stimulation. (A). Human neural cells cultured on poly-D-lysine pre-coated 96 well plates were pretreated with supernatants from non-activated CD8 T cells (Unstim.), anti-CD3 or anti-CD3/CD28-activated CD8+ T cells without MgTx (none), and with MgTx (MgTx). After 24 hours of treatment, CellQuanti-blue dye was added in each well for 30 minutes. Fluorescence was then detected using a plate reader. Cell viability was quantified by fluorescence intensity. (B). MgTx (30 nM) was added to culture media and incubated for 3 days. Human NPCs were treated with supernatants without MgTx (Ctrl), MgTx contained sups with vehicle treatment, with GrB alone (GrB) or MgTx containing sups plus GrB treatment (GrB/MgTx sups). Neurotoxicity was evaluated by cell viability quantified by fluorescence intensity. The fluorescence intensity in each group is plotted as percent relative to that in non-activated cells (Unstim.) or control cells (Ctrl). Data are mean of triplicate ± SD of one representative of three independent experiments. Values that are significantly different from that of vehicle treated control are indicated as *, p <0.05; **, p <0.01; ***, p <0.005.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Cell Culture, Fluorescence, Incubation

Journal: PLoS ONE

Article Title: Next Generation Inactivated Polio Vaccine Manufacturing to Support Post Polio-Eradication Biosafety Goals

doi: 10.1371/journal.pone.0083374

Figure Lengend Snippet: Biosafety and viral safety testing of Sabin PV master (MS) and working (WS) seedlots.

Article Snippet: Sabin PV type 1 (LSc 2ab KP 2 ), Sabin PV type 2 (P712 Ch2ab-KP 2 ) and Sabin PV type 3 (Lot 457-III-Pfizer) were used.

Techniques:

Journal: PLoS ONE

Article Title: Next Generation Inactivated Polio Vaccine Manufacturing to Support Post Polio-Eradication Biosafety Goals

doi: 10.1371/journal.pone.0083374

Figure Lengend Snippet: Product recovery during processing of two batches for each serotype.

Article Snippet: Sabin PV type 1 (LSc 2ab KP 2 ), Sabin PV type 2 (P712 Ch2ab-KP 2 ) and Sabin PV type 3 (Lot 457-III-Pfizer) were used.

Techniques: Clarification Assay, Concentration Assay