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naïve cd4 t cell isolation kit  (Miltenyi Biotec)


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    Structured Review

    Miltenyi Biotec naïve cd4 t cell isolation kit
    Naïve Cd4 T Cell Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+na%C3%AFve+cd4+t+cells+isolation+kit/Naive+CD4%2B+T+Cell+Isolation+Kit%2C+mouse/pmc13427790-70-17-25
    Average 97 stars, based on 435 article reviews
    naïve cd4 t cell isolation kit - by Bioz Stars, 2026-09
    97/100 stars

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    Related Articles

    Purification:

    Article Title: Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Dextran Sodium Sulfate-Induced Ulcerative Colitis
    Article Snippet: .. Total spleen T cells were purified by negative selection with the mouse naïve CD4 + T Cells Isolation Kit (Miltenyi Biotec, Cat# 130-104-453) and LS separation columns (Miltenyi Biotec, Cat# 130-042-401) following the manufacturer’s instructions. .. Naive CD4 + T cells were seeded in 96-well plates coated with anti-CD3 (Thermo Fisher Scientific, Cat# 16-0032-85) and anti-CD28 (Thermo Fisher Scientific, Cat# 16-0281-85) antibodies at 2 x 10 5 cells/well in RPMI 1640 medium (Gibco, Cat# 11875-119) containing 10% inactivated fetal bovine serum (Gibco, Cat# 10099141).

    Article Title: EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice
    Article Snippet: .. Naïve CD4 + T cells were purified from the splenocytes of 6-7-week-old EGR2 -/- B6/lpr and control mice by using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec as we previously reported ( ). .. For Th1 differentiation, naïve CD4 + T cells (1.5x10 6 ) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X cell, Lebanon, NH, USA), 1 μg/ml soluble anti-CD28 (clone 37.51, Bio X cell), 5ng/ml IL-2 (eBioscience), 10ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL4 (Bio X cell) for 3 days.

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells.
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4+ T cells were purified from the splenocytes of 6–8-week-old MRL and MRL-lpr mice by negative selection using a mouse naïve CD4+T cells isolation kit from Miltenyi Biotec. ..

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4 + T cells were purified from the splenocytes of 6–8-week-old MRL and MRL- lpr mice by negative selection using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec. ..

    Selection:

    Article Title: Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Dextran Sodium Sulfate-Induced Ulcerative Colitis
    Article Snippet: .. Total spleen T cells were purified by negative selection with the mouse naïve CD4 + T Cells Isolation Kit (Miltenyi Biotec, Cat# 130-104-453) and LS separation columns (Miltenyi Biotec, Cat# 130-042-401) following the manufacturer’s instructions. .. Naive CD4 + T cells were seeded in 96-well plates coated with anti-CD3 (Thermo Fisher Scientific, Cat# 16-0032-85) and anti-CD28 (Thermo Fisher Scientific, Cat# 16-0281-85) antibodies at 2 x 10 5 cells/well in RPMI 1640 medium (Gibco, Cat# 11875-119) containing 10% inactivated fetal bovine serum (Gibco, Cat# 10099141).

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells.
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4+ T cells were purified from the splenocytes of 6–8-week-old MRL and MRL-lpr mice by negative selection using a mouse naïve CD4+T cells isolation kit from Miltenyi Biotec. ..

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4 + T cells were purified from the splenocytes of 6–8-week-old MRL and MRL- lpr mice by negative selection using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec. ..

    Isolation:

    Article Title: Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Dextran Sodium Sulfate-Induced Ulcerative Colitis
    Article Snippet: .. Total spleen T cells were purified by negative selection with the mouse naïve CD4 + T Cells Isolation Kit (Miltenyi Biotec, Cat# 130-104-453) and LS separation columns (Miltenyi Biotec, Cat# 130-042-401) following the manufacturer’s instructions. .. Naive CD4 + T cells were seeded in 96-well plates coated with anti-CD3 (Thermo Fisher Scientific, Cat# 16-0032-85) and anti-CD28 (Thermo Fisher Scientific, Cat# 16-0281-85) antibodies at 2 x 10 5 cells/well in RPMI 1640 medium (Gibco, Cat# 11875-119) containing 10% inactivated fetal bovine serum (Gibco, Cat# 10099141).

    Article Title: EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice
    Article Snippet: .. Naïve CD4 + T cells were purified from the splenocytes of 6-7-week-old EGR2 -/- B6/lpr and control mice by using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec as we previously reported ( ). .. For Th1 differentiation, naïve CD4 + T cells (1.5x10 6 ) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X cell, Lebanon, NH, USA), 1 μg/ml soluble anti-CD28 (clone 37.51, Bio X cell), 5ng/ml IL-2 (eBioscience), 10ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL4 (Bio X cell) for 3 days.

    Article Title: TNF‐ α ‐Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy‐Related Protein VCP
    Article Snippet: Red blood cells (RBCs) were lysed by incubating the cells in RBC lysis buffer (Solarbio, #R1010) for 15 min at 4 °C. .. Splenocytes were then collected by centrifugation at 1500 rpm for 5 min. For the isolation of naïve CD4+ T cells in the spleen, a Mouse Naïve CD4+ T Cells Isolation Kit (Miltenyi Biotec, #130‐104‐453) was used according to the manufacturer's protocol. ..

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells.
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4+ T cells were purified from the splenocytes of 6–8-week-old MRL and MRL-lpr mice by negative selection using a mouse naïve CD4+T cells isolation kit from Miltenyi Biotec. ..

    Article Title: EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4 + T cells
    Article Snippet: The flow data were analyzed with FlowJo version 10 software. .. Naïve CD4 + T cells were purified from the splenocytes of 6–8-week-old MRL and MRL- lpr mice by negative selection using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec. ..

    Article Title: An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory.
    Article Snippet: .. Naïve CD45.2 CD4+ OT-II CD4+ T cells were isolated from OT-II mice (C57BL/6-Tg(TcraTcrb)425Cbn/Cr; Charles River) using mouse naïve CD4+ T cells isolation kit (130-104-452, Miltenyi). .. Naïve CD45.2 OT-II CD4+ T cells were then coupled with congenic mouse CD45.2 APCs (CD3-depleted splenocytes, by CD3e Microbeads kit mouse 130-094-973, Miltenyi) that were previously live labelled with Cy3–TelC probes, in the presence of OVA antigen (3 μM).

    Article Title: TNF-α-Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy-Related Protein VCP.
    Article Snippet: Red blood cells (RBCs) were lysed by incubating the cells in RBC lysis buffer (Solarbio, #R1010) for 15 min at 4 °C. .. Splenocytes were then collected by centrifugation at 1500 rpm for 5 min. For the isolation of naïve CD4+ T cells in the spleen, a Mouse Naïve CD4+ T Cells Isolation Kit (Miltenyi Biotec, #130-104-453) was used according to the manufacturer’s protocol. ..

    Control:

    Article Title: EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice
    Article Snippet: .. Naïve CD4 + T cells were purified from the splenocytes of 6-7-week-old EGR2 -/- B6/lpr and control mice by using a mouse naïve CD4 + T cells isolation kit from Miltenyi Biotec as we previously reported ( ). .. For Th1 differentiation, naïve CD4 + T cells (1.5x10 6 ) were plated in 48-well plate and cultured with 2 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X cell, Lebanon, NH, USA), 1 μg/ml soluble anti-CD28 (clone 37.51, Bio X cell), 5ng/ml IL-2 (eBioscience), 10ng/ml IL-12 (eBioscience) and 10 μg/ml anti-IL4 (Bio X cell) for 3 days.

    Centrifugation:

    Article Title: TNF‐ α ‐Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy‐Related Protein VCP
    Article Snippet: Red blood cells (RBCs) were lysed by incubating the cells in RBC lysis buffer (Solarbio, #R1010) for 15 min at 4 °C. .. Splenocytes were then collected by centrifugation at 1500 rpm for 5 min. For the isolation of naïve CD4+ T cells in the spleen, a Mouse Naïve CD4+ T Cells Isolation Kit (Miltenyi Biotec, #130‐104‐453) was used according to the manufacturer's protocol. ..

    Article Title: TNF-α-Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy-Related Protein VCP.
    Article Snippet: Red blood cells (RBCs) were lysed by incubating the cells in RBC lysis buffer (Solarbio, #R1010) for 15 min at 4 °C. .. Splenocytes were then collected by centrifugation at 1500 rpm for 5 min. For the isolation of naïve CD4+ T cells in the spleen, a Mouse Naïve CD4+ T Cells Isolation Kit (Miltenyi Biotec, #130-104-453) was used according to the manufacturer’s protocol. ..



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    GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl <t>CD4</t> Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.
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    Expected results of this protocol <t>Naïve</t> CD4 + T cells cultured for d5 in supernatant of untreated BMDCs (SN NT) or in supernatant of BPI-treated BMDCs (SN BPI). (A) Representative dot blot of flow cytometric analysis of CD62L and CD44 cell surface presentation. (B) IL-22 secretion measured by Luminex technology, n = 4. Data are shown as means ± SEM. Statistical testing was performed using Student`s ratio paired t test.
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    Image Search Results


    GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

    Journal: Bioactive Materials

    Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

    doi: 10.1016/j.bioactmat.2026.02.039

    Figure Lengend Snippet: GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

    Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

    Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Generated, Flow Cytometry, Comparison

    GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

    Journal: Bioactive Materials

    Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

    doi: 10.1016/j.bioactmat.2026.02.039

    Figure Lengend Snippet: GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

    Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

    Techniques: Isolation, Flow Cytometry, Expressing, Comparison

    Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ p < 0.05.

    Journal: Bioactive Materials

    Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

    doi: 10.1016/j.bioactmat.2026.02.039

    Figure Lengend Snippet: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ p < 0.05.

    Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

    Techniques: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

    BOLT activates T cell PI3K-AKT-mTOR pathway to enhance T cell anti-tumor effect. (A) Flow cytometry plots compare IFN-γ and IL-2 expression at pH 7.8 and 6.0 along with various doses of BOLT in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (B, C) Bar graphs show IFN-γ and IL-2 expression in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (D) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 antibodies and incubated for 3 days with cell culture media of different pH levels. Western blot was performed to determine the phosphorylation of Akt and S6 under acidic conditions (pH 6.5) and alkaline pH (7.8). (E) Activated CD4 + T cells were treated with 0, 0.25, and 0.5 mg/mL doses of BOLT following CD4 + T cells activation at pH 7.8. Western blot analysis showing the phosphorylation of Akt and S6 were observed. (F) CD4 + T cells were activated and treated with BOLT at acidic pH. Western blot analysis was performed to determine the phosphorylation of Akt and S6. Two-way ANOVA was used for multiple comparisons. Experiments were conducted in triplicate. Data are mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

    Journal: Bioactive Materials

    Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

    doi: 10.1016/j.bioactmat.2026.02.039

    Figure Lengend Snippet: BOLT activates T cell PI3K-AKT-mTOR pathway to enhance T cell anti-tumor effect. (A) Flow cytometry plots compare IFN-γ and IL-2 expression at pH 7.8 and 6.0 along with various doses of BOLT in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (B, C) Bar graphs show IFN-γ and IL-2 expression in CD4 + T cells from CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (D) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 antibodies and incubated for 3 days with cell culture media of different pH levels. Western blot was performed to determine the phosphorylation of Akt and S6 under acidic conditions (pH 6.5) and alkaline pH (7.8). (E) Activated CD4 + T cells were treated with 0, 0.25, and 0.5 mg/mL doses of BOLT following CD4 + T cells activation at pH 7.8. Western blot analysis showing the phosphorylation of Akt and S6 were observed. (F) CD4 + T cells were activated and treated with BOLT at acidic pH. Western blot analysis was performed to determine the phosphorylation of Akt and S6. Two-way ANOVA was used for multiple comparisons. Experiments were conducted in triplicate. Data are mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

    Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

    Techniques: Flow Cytometry, Expressing, Incubation, Cell Culture, Western Blot, Phospho-proteomics, Activation Assay

    Expected results of this protocol Naïve CD4 + T cells cultured for d5 in supernatant of untreated BMDCs (SN NT) or in supernatant of BPI-treated BMDCs (SN BPI). (A) Representative dot blot of flow cytometric analysis of CD62L and CD44 cell surface presentation. (B) IL-22 secretion measured by Luminex technology, n = 4. Data are shown as means ± SEM. Statistical testing was performed using Student`s ratio paired t test.

    Journal: STAR Protocols

    Article Title: Protocol for potent activation of T cells using BPI-stimulated murine bone marrow-derived cells

    doi: 10.1016/j.xpro.2026.104519

    Figure Lengend Snippet: Expected results of this protocol Naïve CD4 + T cells cultured for d5 in supernatant of untreated BMDCs (SN NT) or in supernatant of BPI-treated BMDCs (SN BPI). (A) Representative dot blot of flow cytometric analysis of CD62L and CD44 cell surface presentation. (B) IL-22 secretion measured by Luminex technology, n = 4. Data are shown as means ± SEM. Statistical testing was performed using Student`s ratio paired t test.

    Article Snippet: Note: If no FACS device is available, the sorting of naïve T cells can be performed using a naïve MACS Sort Kit from Miltenyi Biotec (130-104-453 ).

    Techniques: Cell Culture, Dot Blot, Luminex