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ionomycin  (InvivoGen)


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    InvivoGen ionomycin
    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + <t>Ionomycin</t> for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.
    Ionomycin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ionomycin/Ionomycin/pmc13223964-74-0-2
    Average 95 stars, based on 108 article reviews
    ionomycin - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "VPS33B regulates MHC class II antigen presentation in dendritic cells to drive CD4 T cell immunity"

    Article Title: VPS33B regulates MHC class II antigen presentation in dendritic cells to drive CD4 T cell immunity

    Journal: iScience

    doi: 10.1016/j.isci.2026.116052

    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.
    Figure Legend Snippet: Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.

    Techniques Used: In Vivo, Injection, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

    Related Articles

    other:

    Article Title: Microbial metabolite indole-3-propionic acid drives mitochondrial respiration in CD4 + T cells to confer protection against intestinal inflammation.
    Article Snippet: The gut microbiota and its metabolites critically regulate immune cell phenotype, function and energy metabolism.. We screened a collection of gut microbiota-related metabolites to identify modulators of mitochondrial metabolism in T cells.. Here we show that indole-3-propionic acid (IPA) stimulates mitochondrial respiration of CD4 T cells by increasing fatty acid oxidation (FAO) and amino acid oxidation (AAO), while inhibiting glycolytic capacity.

    Transfection:

    Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein
    Article Snippet: .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega). .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

    Incubation:

    Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein
    Article Snippet: .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega). .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

    Luciferase:

    Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein
    Article Snippet: .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega). .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

    Lysis:

    Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein
    Article Snippet: .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega). .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

    Activity Assay:

    Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein
    Article Snippet: .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega). .. 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

    Positive Control:

    Article Title: Highly focused human CD8+ T-cell response in the lower airways during acute influenza infection.
    Article Snippet: .. Positive control phorbol 12-myristate 13-acetate (InvivoGen) and ionomycin (InvivoGen) were added to a separate well. .. Positive control phorbol 12-myristate 13-acetate (InvivoGen) and ionomycin (InvivoGen) were added to a separate well.

    Control:

    Article Title: MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity
    Article Snippet: .. Following adherence, neutrophils were stimulated with either vehicle control, 4 μM ionomycin (Invivogen), or 100nM PMA (Invivogen) for 4 hours at 37°C and 5% CO2. ..



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    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + <t>Ionomycin</t> for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.
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    Yeasen Biotechnology 11201es03 ionomycin yeasen
    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + <t>Ionomycin</t> for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.
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    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + <t>Ionomycin</t> for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.
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    Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.

    Journal: iScience

    Article Title: VPS33B regulates MHC class II antigen presentation in dendritic cells to drive CD4 T cell immunity

    doi: 10.1016/j.isci.2026.116052

    Figure Lengend Snippet: Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.

    Article Snippet: Ionomycin , Invivogen , Cat#inh-ion.

    Techniques: In Vivo, Injection, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay