protease k 2  (Millipore)


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    Structured Review

    Millipore protease k 2
    Protease K 2, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease k 2/product/Millipore
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protease k 2 - by Bioz Stars, 2020-03
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    tissue permeabilisation

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    Incubation:

    Article Title: Prostate-specific PTen deletion in mice activates inflammatory microRNA expression pathways in the epithelium early in hyperplasia development
    Article Snippet: Tissue permeabilisation was performed using Protease-K 2–15 μg/ml (Sigma-Aldrich, UK). .. Slides were washed in diethyl pyrocarbonate (DEPC) water, and incubated in prehybridisation solution for 60 mins at 37 °C, then an equal volume of miR probe (dig-labelled miR155/21/scrambled—250 ng/μl), and incubated overnight.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Prostate-specific PTen deletion in mice activates inflammatory microRNA expression pathways in the epithelium early in hyperplasia development
    Article Snippet: In situ hybridisation FFPE sections (5 μm), were dewaxed and re-hydrated. .. Tissue permeabilisation was performed using Protease-K 2–15 μg/ml (Sigma-Aldrich, UK).

    In Situ:

    Article Title: Prostate-specific PTen deletion in mice activates inflammatory microRNA expression pathways in the epithelium early in hyperplasia development
    Article Snippet: Paragraph title: In situ hybridisation ... Tissue permeabilisation was performed using Protease-K 2–15 μg/ml (Sigma-Aldrich, UK).

    Hybridization:

    Article Title: Prostate-specific PTen deletion in mice activates inflammatory microRNA expression pathways in the epithelium early in hyperplasia development
    Article Snippet: Paragraph title: In situ hybridisation ... Tissue permeabilisation was performed using Protease-K 2–15 μg/ml (Sigma-Aldrich, UK).

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    Millipore cathepsin k inhibitor ii
    Effect of <t>cathepsin</t> K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P
    Cathepsin K Inhibitor Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cathepsin k inhibitor ii/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore granzyme b
    Production of perforin, <t>granzyme</t> B and granulysin in CD8 + T cells as well as CD4 + T cells. ( A ) Enhanced production of perforin and granzyme B from CD8 + T cells cultured with LipoK stimulated M. leprae infected DCs. Intracellular staining of perforin and granzyme B was performed as follows: Cells were first stained with PE conjugated anti-CD4 or APC conjugated anti-CD8 mAb. Then, the cells were fixed in 2% formaldehyde, permeabilized in 0.1% saponin, and stained with FITC conjugated anti-perforin mAb or anti-granzyme B mAb or isotype control IgG2a. Figure shows the dot plot of the gated CD8 + T cells. The right hand quadrant shows CD8 high T cells (activated CD8 + T cells) and the number indicates the percentage of perforin or granzyme B positive T cells among gated CD8 high T cells. *To determine whether direct interaction between CD4 + and CD8 + T cells for perforin and granzyme B production from CD8 + T cells, is needed, CD4 + T cells were cultured in inserts in a 24-well plate, and were not allowed to interact directly with CD8 + T cells. As a control experiment, exogenous IL-2 (in the left hand dot plot) at a concentration of 50 U/ml was added to CD8 + T cells. ( B ) Enhanced expression of perforin and granzyme B from CD4 + T cells. The right hand quadrant shows CD4 high T cells, and the number indicates percentage of CD4 high T cells producing perforin and granzyme B. ( C ) Enhanced expression of granulysin from CD8 + and CD4 + T cells, co-cultured with LipoK and M. leprae stimulated DCs. The protocol was followed as per the staining of perforin, except that the surface stain used was FITC conjugated-CD4 and APC conjugated anti-CD8 mAb, and subsequently PE conjugated granulysin was used. Figure shows the dot plot of the gated CD8 + and CD4 + T cells. The right hand quadrant shows CD8 high or CD4 high T cells (activated T cells) and the number indicates the percentage of granulysin positive T cells among gated CD8 high and CD4 high T cells. Representative data of three separate experiments with different donors is shown.
    Granzyme B, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/granzyme b/product/Millipore
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    Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Isolation, Mouse Assay

    Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay

    A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Staining, Quantitation Assay, Aqueous Normal-phase Chromatography, Isolation

    Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Isolation

    Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Expressing, Knock-Out, Mouse Assay, Fluorescence, Western Blot, Quantitation Assay

    A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Cell Culture, Labeling, Knock-Out, Mouse Assay, Isolation, Western Blot

    Production of perforin, granzyme B and granulysin in CD8 + T cells as well as CD4 + T cells. ( A ) Enhanced production of perforin and granzyme B from CD8 + T cells cultured with LipoK stimulated M. leprae infected DCs. Intracellular staining of perforin and granzyme B was performed as follows: Cells were first stained with PE conjugated anti-CD4 or APC conjugated anti-CD8 mAb. Then, the cells were fixed in 2% formaldehyde, permeabilized in 0.1% saponin, and stained with FITC conjugated anti-perforin mAb or anti-granzyme B mAb or isotype control IgG2a. Figure shows the dot plot of the gated CD8 + T cells. The right hand quadrant shows CD8 high T cells (activated CD8 + T cells) and the number indicates the percentage of perforin or granzyme B positive T cells among gated CD8 high T cells. *To determine whether direct interaction between CD4 + and CD8 + T cells for perforin and granzyme B production from CD8 + T cells, is needed, CD4 + T cells were cultured in inserts in a 24-well plate, and were not allowed to interact directly with CD8 + T cells. As a control experiment, exogenous IL-2 (in the left hand dot plot) at a concentration of 50 U/ml was added to CD8 + T cells. ( B ) Enhanced expression of perforin and granzyme B from CD4 + T cells. The right hand quadrant shows CD4 high T cells, and the number indicates percentage of CD4 high T cells producing perforin and granzyme B. ( C ) Enhanced expression of granulysin from CD8 + and CD4 + T cells, co-cultured with LipoK and M. leprae stimulated DCs. The protocol was followed as per the staining of perforin, except that the surface stain used was FITC conjugated-CD4 and APC conjugated anti-CD8 mAb, and subsequently PE conjugated granulysin was used. Figure shows the dot plot of the gated CD8 + and CD4 + T cells. The right hand quadrant shows CD8 high or CD4 high T cells (activated T cells) and the number indicates the percentage of granulysin positive T cells among gated CD8 high and CD4 high T cells. Representative data of three separate experiments with different donors is shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

    doi: 10.1371/journal.pntd.0001401

    Figure Lengend Snippet: Production of perforin, granzyme B and granulysin in CD8 + T cells as well as CD4 + T cells. ( A ) Enhanced production of perforin and granzyme B from CD8 + T cells cultured with LipoK stimulated M. leprae infected DCs. Intracellular staining of perforin and granzyme B was performed as follows: Cells were first stained with PE conjugated anti-CD4 or APC conjugated anti-CD8 mAb. Then, the cells were fixed in 2% formaldehyde, permeabilized in 0.1% saponin, and stained with FITC conjugated anti-perforin mAb or anti-granzyme B mAb or isotype control IgG2a. Figure shows the dot plot of the gated CD8 + T cells. The right hand quadrant shows CD8 high T cells (activated CD8 + T cells) and the number indicates the percentage of perforin or granzyme B positive T cells among gated CD8 high T cells. *To determine whether direct interaction between CD4 + and CD8 + T cells for perforin and granzyme B production from CD8 + T cells, is needed, CD4 + T cells were cultured in inserts in a 24-well plate, and were not allowed to interact directly with CD8 + T cells. As a control experiment, exogenous IL-2 (in the left hand dot plot) at a concentration of 50 U/ml was added to CD8 + T cells. ( B ) Enhanced expression of perforin and granzyme B from CD4 + T cells. The right hand quadrant shows CD4 high T cells, and the number indicates percentage of CD4 high T cells producing perforin and granzyme B. ( C ) Enhanced expression of granulysin from CD8 + and CD4 + T cells, co-cultured with LipoK and M. leprae stimulated DCs. The protocol was followed as per the staining of perforin, except that the surface stain used was FITC conjugated-CD4 and APC conjugated anti-CD8 mAb, and subsequently PE conjugated granulysin was used. Figure shows the dot plot of the gated CD8 + and CD4 + T cells. The right hand quadrant shows CD8 high or CD4 high T cells (activated T cells) and the number indicates the percentage of granulysin positive T cells among gated CD8 high and CD4 high T cells. Representative data of three separate experiments with different donors is shown.

    Article Snippet: When LipoK and M. leprae stimulated DCs were co-cultured with T cells, 12.7% of CD4high T cells produced perforin and 14.6% of those cells produced granzyme B, whereas in presence of M. leprae stimulated DCs, 6.6% produced perforin and 8.3% produced granzyme B ( ).

    Techniques: Cell Culture, Infection, Staining, Concentration Assay, Expressing

    Reduction in the viability of M. leprae in DCs after co-culture with T cells and LipoK stimulation. ( A ) DCs were infected with M. leprae and stimulated with LipoK, 2 days later, cells were collected and the viability of M. leprae in DCs was measured by the radiorespirometric assay (metabolic CO 2 release) as described in Materials and Methods . In brief, 14 C labeled palmitic acid was added to the lysates of DCs and cultured at 33°C. After 7 days of culture, the amount of 14 CO 2 evolved was measured using a Packard 1500 TRI-CARB liquid scintillation analyzer. ( B ) DCs were infected with M. leprae as in A , and co-cultured with T cells. Six days after the co-culture, DCs were lysed, and the viability of M. leprae was determined by the radiorespirometric assay. ( C ) M. leprae at a concentration of 1×10 7 /well/200 µl in Middlebrook 7H9 media was incubated with granulysin or granzyme B for a period of 3 days at 33°C, and the viability determined as described in A . Unpaired Student's t test was used to find the statistical significance of the two sets of data. Representative data of three separate experiments is shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

    doi: 10.1371/journal.pntd.0001401

    Figure Lengend Snippet: Reduction in the viability of M. leprae in DCs after co-culture with T cells and LipoK stimulation. ( A ) DCs were infected with M. leprae and stimulated with LipoK, 2 days later, cells were collected and the viability of M. leprae in DCs was measured by the radiorespirometric assay (metabolic CO 2 release) as described in Materials and Methods . In brief, 14 C labeled palmitic acid was added to the lysates of DCs and cultured at 33°C. After 7 days of culture, the amount of 14 CO 2 evolved was measured using a Packard 1500 TRI-CARB liquid scintillation analyzer. ( B ) DCs were infected with M. leprae as in A , and co-cultured with T cells. Six days after the co-culture, DCs were lysed, and the viability of M. leprae was determined by the radiorespirometric assay. ( C ) M. leprae at a concentration of 1×10 7 /well/200 µl in Middlebrook 7H9 media was incubated with granulysin or granzyme B for a period of 3 days at 33°C, and the viability determined as described in A . Unpaired Student's t test was used to find the statistical significance of the two sets of data. Representative data of three separate experiments is shown.

    Article Snippet: When LipoK and M. leprae stimulated DCs were co-cultured with T cells, 12.7% of CD4high T cells produced perforin and 14.6% of those cells produced granzyme B, whereas in presence of M. leprae stimulated DCs, 6.6% produced perforin and 8.3% produced granzyme B ( ).

    Techniques: Co-Culture Assay, Infection, Labeling, Cell Culture, Concentration Assay, Incubation

    DV CaPNP/multipeptide formulations stimulate CD8+ T cell activation in vitro : (A) IFN-γ ELISpot assay: Using peripheral blood from healthy donors' PBMCs were stimulated in vitro with: 1) pooled peptides, or 2) CaPNP/multipeptide formulation with GlcNAc, or 3) CaPNP/multipeptide formulation without GlcNAc. The activated PBMCs containing the epitope specific CTLs were harvested, washed, and co-cultured overnight with HepG2 targets that were either pulsed with individual peptides (2µg each peptide/mL) or infected with DV2 in an IFN-γ ELISpot assay. Data represented as mean ± S.D (n = 3) of SFU per 1 million PBMCs. (B) Cytolytic response: Identical cultures were set up as in (A) in 96 well round bottom plates. The next day supernatant was harvested and used to detect cytolytic molecules (Granzyme B) using Luminex magnetic bead technology (MagPix). Data represented as mean ± S.D (n = 3) pg/mL. (C) Activation marker analysis: The cells were stained with CD8 and CD107a antibody at the end of the assay culture period to analyze the CTLs for the expression of degranulation marker, CD107a, by flow cytometry. Data represented as mean ± SD (n = 3) percent double positive (both CD8 + and CD107a + staining) cells.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: A novel immunization approach for dengue infection based on conserved T cell epitopes formulated in calcium phosphate nanoparticles

    doi: 10.1080/21645515.2017.1369639

    Figure Lengend Snippet: DV CaPNP/multipeptide formulations stimulate CD8+ T cell activation in vitro : (A) IFN-γ ELISpot assay: Using peripheral blood from healthy donors' PBMCs were stimulated in vitro with: 1) pooled peptides, or 2) CaPNP/multipeptide formulation with GlcNAc, or 3) CaPNP/multipeptide formulation without GlcNAc. The activated PBMCs containing the epitope specific CTLs were harvested, washed, and co-cultured overnight with HepG2 targets that were either pulsed with individual peptides (2µg each peptide/mL) or infected with DV2 in an IFN-γ ELISpot assay. Data represented as mean ± S.D (n = 3) of SFU per 1 million PBMCs. (B) Cytolytic response: Identical cultures were set up as in (A) in 96 well round bottom plates. The next day supernatant was harvested and used to detect cytolytic molecules (Granzyme B) using Luminex magnetic bead technology (MagPix). Data represented as mean ± S.D (n = 3) pg/mL. (C) Activation marker analysis: The cells were stained with CD8 and CD107a antibody at the end of the assay culture period to analyze the CTLs for the expression of degranulation marker, CD107a, by flow cytometry. Data represented as mean ± SD (n = 3) percent double positive (both CD8 + and CD107a + staining) cells.

    Article Snippet: Activated PBMCs cytokine secretion was measured using a Milliplex magnetic bead assay customized to detect Granzyme-B for T cell characterization (Millipore) as described before.

    Techniques: Activation Assay, In Vitro, Enzyme-linked Immunospot, Cell Culture, Infection, Luminex, Marker, Staining, Expressing, Flow Cytometry, Cytometry