Review



buffer flow cytometry permeabilization wash buffer i  (R&D Systems)


Bioz Verified Symbol R&D Systems is a verified supplier
Bioz Manufacturer Symbol R&D Systems manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    R&D Systems buffer flow cytometry permeabilization wash buffer i
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Buffer Flow Cytometry Permeabilization Wash Buffer I, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/buffer flow cytometry permeabilization wash buffer i/product/R&D Systems
    Average 93 stars, based on 111 article reviews
    buffer flow cytometry permeabilization wash buffer i - by Bioz Stars, 2026-03
    93/100 stars

    Images

    1) Product Images from "Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy"

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    Journal: Science Advances

    doi: 10.1126/sciadv.adq3940

    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow cytometry examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Figure Legend Snippet: ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow cytometry examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.

    Techniques Used: Flow Cytometry, Inhibition, Membrane, Fluorescence, Incubation, Labeling, Binding Assay

    ( A ) Schematic illustration of IFN-γ–induced PD-L1 up-regulation on the surface of tumor cells in vitro. ( B to E ) Flow cytometry and Western blot examination of clickable PD-L1 inhibitor–mediated PD-L1 degradation on the surface of tumor cell membrane in vitro. [(B) and (C)] Flow cytometry detection of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (B) and B16-F10 (C) tumor cells. [(D) and (E)] Western blot analysis of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (D) and B16-F10 (E) tumor cells. (F) Representative CLSM images of PD-L1 abundance on the membrane surface of 4T1 tumor cells (scale bar = 20 μm). Na + ,K + -ATPase, Na + - and K + -dependent adenosine triphosphatase. ( G to J ) Flow cytometry–determined PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (G) and B16-F10 (H) tumor cells after treatment with 10 μM BMS-1 or D5B. Western blot analysis and semi-quantification of PD-L1 expression on the membrane surface of IFN-γ–pretreated 4T1 (I) and B16-F10 (J) tumor cells with or without azide labeling. ( K ) Representative flow cytometry plots and quantification of IFN-γ + CD8 + T cells. 1#, CD8 + T cells incubated with BMS-1–treated tumor cells; 2#, CD8 + T cells incubated with D5B-treated tumor cells; 3#, CD8 + T cells incubated with PBS. ( L ) Mechanism illustration for PD-L1 degradation increased proliferation of CD8 + T lymphocytes. The data were presented as the means ± SD. P values were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test [(D) and (E)], unpaired Student’s t test [(I) and (J)], or one-way ANOVA with Tukey’s multiple comparisons test (L). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
    Figure Legend Snippet: ( A ) Schematic illustration of IFN-γ–induced PD-L1 up-regulation on the surface of tumor cells in vitro. ( B to E ) Flow cytometry and Western blot examination of clickable PD-L1 inhibitor–mediated PD-L1 degradation on the surface of tumor cell membrane in vitro. [(B) and (C)] Flow cytometry detection of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (B) and B16-F10 (C) tumor cells. [(D) and (E)] Western blot analysis of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (D) and B16-F10 (E) tumor cells. (F) Representative CLSM images of PD-L1 abundance on the membrane surface of 4T1 tumor cells (scale bar = 20 μm). Na + ,K + -ATPase, Na + - and K + -dependent adenosine triphosphatase. ( G to J ) Flow cytometry–determined PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (G) and B16-F10 (H) tumor cells after treatment with 10 μM BMS-1 or D5B. Western blot analysis and semi-quantification of PD-L1 expression on the membrane surface of IFN-γ–pretreated 4T1 (I) and B16-F10 (J) tumor cells with or without azide labeling. ( K ) Representative flow cytometry plots and quantification of IFN-γ + CD8 + T cells. 1#, CD8 + T cells incubated with BMS-1–treated tumor cells; 2#, CD8 + T cells incubated with D5B-treated tumor cells; 3#, CD8 + T cells incubated with PBS. ( L ) Mechanism illustration for PD-L1 degradation increased proliferation of CD8 + T lymphocytes. The data were presented as the means ± SD. P values were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test [(D) and (E)], unpaired Student’s t test [(I) and (J)], or one-way ANOVA with Tukey’s multiple comparisons test (L). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Techniques Used: In Vitro, Flow Cytometry, Western Blot, Membrane, Expressing, Labeling, Incubation

    ( A ) Acid-responsive mechanism illustration of the pH e -activatable PCP@D5B, pH i -activatable PDP@D5B, and pH-inactivated PBP@D5B nanoparticles for acid-triggered nanoparticle dissociation and activation of NIRF/MRI signals. ( B ) Dynamic light scattering (DLS)– and transmission electron microscopy (TEM)–determined particle size distribution and morphology change of the PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles at pH 7.4 and 6.5, respectively (scale bars, 100 nm). ( C ) Representative CLSM images of cell membrane [wheat germ agglutinin (WGA), green] and colocalization with PPa-labeled nanoparticles (red) at the predesignated time points. 4T1 tumor cells were incubated with PCP@D5B, PDP@D5B, or PBP@D5B nanoparticles for 5 min under different pH conditions (scale bars, 20 μm). ( D ) Schematic illustration for PCP@D5B-performed PD-L1 degradation on the surface of tumor cell membrane by specifically releasing D5B payload at the extracellular acidic microenvironment in vitro. ( E ) Flow cytometry analysis of PD-L1 abundance on the surface of 4T1 tumor cell membrane; inset number represents the MFI values. ( F ) The mechanism of pH-activated NIRF and MRI signals of PCPGd@D5B nanoparticles. ( G ) Representative T 1 maps of PCPGd@D5B nanoparticles determined at varied pH values. ( H ) The longitudinal relaxation rate ( r 1 ) versus Gd 3+ concentration determined at different pH values. ( I ) MRI of 4T1 tumor-bearing mice in vivo. The mice were intravenously injected with PBS or PCPGd@D5B nanoparticles at a Gd 3+ dose of 1.5 mg/kg and then imaged at the predetermined intervals (white circles represent the tumors). The data are presented as the means ± SD. h, hours.
    Figure Legend Snippet: ( A ) Acid-responsive mechanism illustration of the pH e -activatable PCP@D5B, pH i -activatable PDP@D5B, and pH-inactivated PBP@D5B nanoparticles for acid-triggered nanoparticle dissociation and activation of NIRF/MRI signals. ( B ) Dynamic light scattering (DLS)– and transmission electron microscopy (TEM)–determined particle size distribution and morphology change of the PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles at pH 7.4 and 6.5, respectively (scale bars, 100 nm). ( C ) Representative CLSM images of cell membrane [wheat germ agglutinin (WGA), green] and colocalization with PPa-labeled nanoparticles (red) at the predesignated time points. 4T1 tumor cells were incubated with PCP@D5B, PDP@D5B, or PBP@D5B nanoparticles for 5 min under different pH conditions (scale bars, 20 μm). ( D ) Schematic illustration for PCP@D5B-performed PD-L1 degradation on the surface of tumor cell membrane by specifically releasing D5B payload at the extracellular acidic microenvironment in vitro. ( E ) Flow cytometry analysis of PD-L1 abundance on the surface of 4T1 tumor cell membrane; inset number represents the MFI values. ( F ) The mechanism of pH-activated NIRF and MRI signals of PCPGd@D5B nanoparticles. ( G ) Representative T 1 maps of PCPGd@D5B nanoparticles determined at varied pH values. ( H ) The longitudinal relaxation rate ( r 1 ) versus Gd 3+ concentration determined at different pH values. ( I ) MRI of 4T1 tumor-bearing mice in vivo. The mice were intravenously injected with PBS or PCPGd@D5B nanoparticles at a Gd 3+ dose of 1.5 mg/kg and then imaged at the predetermined intervals (white circles represent the tumors). The data are presented as the means ± SD. h, hours.

    Techniques Used: Activation Assay, Transmission Assay, Electron Microscopy, Membrane, Labeling, Incubation, In Vitro, Flow Cytometry, Concentration Assay, In Vivo, Injection

    ( A ) Schematic illustration of pH-triggered extracellular delivery of D5B for PD-L1 degradation. ( B ) Representative IVIS fluorescence images of 4T1 tumor-bearing BALB/c mice in vivo. ( C ) Semiquantitative of PPa fluorescence intensity from (B) ( n = 3 mice). ( D ) High-performance liquid chromatography (HPLC)–determined pharmacokinetics of D5B-loaded PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles or free D5B ( n = 3 mice). ( E ) HPLC-determined D5B distribution in the tumor mass after intravenous injection ( n = 3 mice). ( F ) Experimental schedule for antitumor study in vivo. it, intratumoral; iv, intravenous; sc, subcutaneous. ( G and H ) Averaged tumor growth curves (G), and (H) animal survival curves of 4T1 tumor-bearing mice (n = 6 mice). ( I and J ) Immunohistochemical (IHC) (I) and flow cytometry (J) examination of PD-L1 abundance 3 days after treatment ( n = 3 mice; scale bars, 50 μm). ( K ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD3 + CD45 + ) ( n = 5 mice). ( L ) Flow cytometry examination of tumor-infiltrating IFN-γ + CD8 + T cells (n = 5 mice). ( M and N ) Tumor mass normalized number of tumor-infiltrating CD8 + (M) and IFN-γ + CD8 + T cells (N) ( n = 5 mice). ( O ) Enzyme-linked immunosorbent assay (ELISA) analysis of intratumoral IFN-γ cytokine secretion at 1, 3, and 7 days after treatment ( n = 3 mice). ( P ) IHC examination of PD-L1 abundance in the normal tissue 3 days after the treatment. ( Q ) Schematic description for tumor-specific delivery of D5B and PD-L1 inhibition with the pH e -activatable nanoparticles. All data are presented as the means ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test [(J) to (N)], repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(E), (G), and (O)], log-rank test (H), or unpaired Student’s t test (P). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.
    Figure Legend Snippet: ( A ) Schematic illustration of pH-triggered extracellular delivery of D5B for PD-L1 degradation. ( B ) Representative IVIS fluorescence images of 4T1 tumor-bearing BALB/c mice in vivo. ( C ) Semiquantitative of PPa fluorescence intensity from (B) ( n = 3 mice). ( D ) High-performance liquid chromatography (HPLC)–determined pharmacokinetics of D5B-loaded PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles or free D5B ( n = 3 mice). ( E ) HPLC-determined D5B distribution in the tumor mass after intravenous injection ( n = 3 mice). ( F ) Experimental schedule for antitumor study in vivo. it, intratumoral; iv, intravenous; sc, subcutaneous. ( G and H ) Averaged tumor growth curves (G), and (H) animal survival curves of 4T1 tumor-bearing mice (n = 6 mice). ( I and J ) Immunohistochemical (IHC) (I) and flow cytometry (J) examination of PD-L1 abundance 3 days after treatment ( n = 3 mice; scale bars, 50 μm). ( K ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD3 + CD45 + ) ( n = 5 mice). ( L ) Flow cytometry examination of tumor-infiltrating IFN-γ + CD8 + T cells (n = 5 mice). ( M and N ) Tumor mass normalized number of tumor-infiltrating CD8 + (M) and IFN-γ + CD8 + T cells (N) ( n = 5 mice). ( O ) Enzyme-linked immunosorbent assay (ELISA) analysis of intratumoral IFN-γ cytokine secretion at 1, 3, and 7 days after treatment ( n = 3 mice). ( P ) IHC examination of PD-L1 abundance in the normal tissue 3 days after the treatment. ( Q ) Schematic description for tumor-specific delivery of D5B and PD-L1 inhibition with the pH e -activatable nanoparticles. All data are presented as the means ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test [(J) to (N)], repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(E), (G), and (O)], log-rank test (H), or unpaired Student’s t test (P). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

    Techniques Used: Fluorescence, In Vivo, High Performance Liquid Chromatography, Injection, Immunohistochemical staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Inhibition

    ( A ) Treatment schedule of in 4T1 tumor model in vivo. ( B ) Individual 4T1 tumor growth curves [complete regression (CR)] ( n = 6 mice). ( C ) Survival rates of 4T1 tumor-bearing mice. ( D ) Flow cytometry analysis of CD86 + CD80 + DCs ( n = 3 mice). ( E ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells, and ( F ) IFN-γ + CD8 + T cells ( n = 5 mice). ( G to I ) Absolute numbers of tumor-infiltrating CD3 + (G), CD8 + (H), and IFN-γ + CD8 + (I) T cells after the indicated treatments ( n = 5 mice). ( J ) M2/M1 ratio after treatment ( n = 5 mice). ( K ) Flow cytometry–determined PD-L1 abundance on the surface of tumor cells membrane ( n = 5 mice). ( L ) Treatment schedule of 4T1 abscopal tumor model (T1 and T2 represents the primary and abscopal tumors, respectively). ( M ) Averaged tumor growth curves ( n = 6 mice), and ( N ) Survival rates of the mice ( n = 6 mice). ( O ) Flow cytometry–determined tumor-infiltrating CD8 + T cells. ( P ) Flow cytometry analysis of T EM cells (CD62L − CD44 + ) in the spleens of 4T1 tumor-bearing mice ( n = 5 mice). ( Q ) Hematoxylin and eosin staining and quantification of metastatic tumor lesions in the lung ( n = 6 mice; scale bars, 2.5 mm). ( R ) Mechanism illustration for combinatory therapy–elicited antitumor immunity and immunological memory to suppress abscopal tumor and lung metastases. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(B) and (M)], log-rank test [(C) and (N)], or one-way ANOVA with Tukey’s post hoc test [(D) to (K) and (O) to (Q)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
    Figure Legend Snippet: ( A ) Treatment schedule of in 4T1 tumor model in vivo. ( B ) Individual 4T1 tumor growth curves [complete regression (CR)] ( n = 6 mice). ( C ) Survival rates of 4T1 tumor-bearing mice. ( D ) Flow cytometry analysis of CD86 + CD80 + DCs ( n = 3 mice). ( E ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells, and ( F ) IFN-γ + CD8 + T cells ( n = 5 mice). ( G to I ) Absolute numbers of tumor-infiltrating CD3 + (G), CD8 + (H), and IFN-γ + CD8 + (I) T cells after the indicated treatments ( n = 5 mice). ( J ) M2/M1 ratio after treatment ( n = 5 mice). ( K ) Flow cytometry–determined PD-L1 abundance on the surface of tumor cells membrane ( n = 5 mice). ( L ) Treatment schedule of 4T1 abscopal tumor model (T1 and T2 represents the primary and abscopal tumors, respectively). ( M ) Averaged tumor growth curves ( n = 6 mice), and ( N ) Survival rates of the mice ( n = 6 mice). ( O ) Flow cytometry–determined tumor-infiltrating CD8 + T cells. ( P ) Flow cytometry analysis of T EM cells (CD62L − CD44 + ) in the spleens of 4T1 tumor-bearing mice ( n = 5 mice). ( Q ) Hematoxylin and eosin staining and quantification of metastatic tumor lesions in the lung ( n = 6 mice; scale bars, 2.5 mm). ( R ) Mechanism illustration for combinatory therapy–elicited antitumor immunity and immunological memory to suppress abscopal tumor and lung metastases. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(B) and (M)], log-rank test [(C) and (N)], or one-way ANOVA with Tukey’s post hoc test [(D) to (K) and (O) to (Q)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Techniques Used: In Vivo, Flow Cytometry, Membrane, Staining

    ( A ) Treatment schedule for the antitumor study in B16-F10 tumor-bearing mice in vivo. ( B ) The averaged and individual B16-F10 tumor growth curves, and ( C ) survival curves of B16-F10 tumor-bearing mice monitored during the therapy period (CR represents the fractions of complete tumor regression at the end of antitumor study, n = 6 or 7 mice). ( D and E ) Representative flow cytometry plots (D), and quantification data of CD86 + CD80 + DCs (E) ( n = 3 mice). ( F and G ) Flow cytometry–determined fractions of (F) M2-phenotype (CD11b + CD206 + ) and (G) M1-phenotype (CD11b + CD80 + ) TAMs ( n = 5 mice). ( H ) The M2/M1 ratio of TAMs. ( I and J ) The MFIs of PD-L1 + TAMs (CD11b + CD80 + ) (I), and PD-L1 + CD45 − tumor cells (J) after treatment. ( K ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs in the tumor sections (scale bars, 40 μm). ( L and M ) Representative flow cytometry plots (L) and quantification (M) of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD45 + CD3 + ) ( n = 5 mice). ( N ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs, and CD8 + T cells in the tumor sections (scale bars, 40 μm). ( O ) Schematic illustration of the clickable PD-L1 inhibitor mitigated the acquired immune evasion. RT induces ITM by up-regulating PD-L1 and recruiting M2-type TAMs, which was reversed with the clickable PD-L1 inhibitor through degrading PD-L1 on the surface of tumor cell membrane and repolarizing M2-type TAMs to M1 type. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test (B), log-rank test (C), or one-way ANOVA with Tukey’s post hoc test [(E) to (J) and (M)], * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
    Figure Legend Snippet: ( A ) Treatment schedule for the antitumor study in B16-F10 tumor-bearing mice in vivo. ( B ) The averaged and individual B16-F10 tumor growth curves, and ( C ) survival curves of B16-F10 tumor-bearing mice monitored during the therapy period (CR represents the fractions of complete tumor regression at the end of antitumor study, n = 6 or 7 mice). ( D and E ) Representative flow cytometry plots (D), and quantification data of CD86 + CD80 + DCs (E) ( n = 3 mice). ( F and G ) Flow cytometry–determined fractions of (F) M2-phenotype (CD11b + CD206 + ) and (G) M1-phenotype (CD11b + CD80 + ) TAMs ( n = 5 mice). ( H ) The M2/M1 ratio of TAMs. ( I and J ) The MFIs of PD-L1 + TAMs (CD11b + CD80 + ) (I), and PD-L1 + CD45 − tumor cells (J) after treatment. ( K ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs in the tumor sections (scale bars, 40 μm). ( L and M ) Representative flow cytometry plots (L) and quantification (M) of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD45 + CD3 + ) ( n = 5 mice). ( N ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs, and CD8 + T cells in the tumor sections (scale bars, 40 μm). ( O ) Schematic illustration of the clickable PD-L1 inhibitor mitigated the acquired immune evasion. RT induces ITM by up-regulating PD-L1 and recruiting M2-type TAMs, which was reversed with the clickable PD-L1 inhibitor through degrading PD-L1 on the surface of tumor cell membrane and repolarizing M2-type TAMs to M1 type. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test (B), log-rank test (C), or one-way ANOVA with Tukey’s post hoc test [(E) to (J) and (M)], * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Techniques Used: In Vivo, Flow Cytometry, Immunofluorescence, Staining, Quantitation Assay, Membrane



    Similar Products

    buffer flow cytometry permeabilization wash buffer i  (R&D Systems)
    93
    R&D Systems buffer flow cytometry permeabilization wash buffer i
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Buffer Flow Cytometry Permeabilization Wash Buffer I, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/buffer flow cytometry permeabilization wash buffer i/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    buffer flow cytometry permeabilization wash buffer i - by Bioz Stars, 2026-03
    93/100 stars
    		  Buy from Supplier
    cells flow cytometry permeabilization wash buffer i  (R&D Systems)
    93
    R&D Systems cells flow cytometry permeabilization wash buffer i
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Cells Flow Cytometry Permeabilization Wash Buffer I, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cells flow cytometry permeabilization wash buffer i/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    cells flow cytometry permeabilization wash buffer i - by Bioz Stars, 2026-03
    93/100 stars
    		  Buy from Supplier
    flow cytometry buffer  (R&D Systems)
    93
    R&D Systems flow cytometry buffer
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Flow Cytometry Buffer, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry buffer/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    flow cytometry buffer - by Bioz Stars, 2026-03
    93/100 stars
    		  Buy from Supplier
    triton x 100  (R&D Systems)
    96
    R&D Systems triton x 100
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Triton X 100, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/R&D Systems
    Average 96 stars, based on 1 article reviews
    triton x 100 - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier
    permeabilization wash buffer i  (R&D Systems)
    96
    R&D Systems permeabilization wash buffer i
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Permeabilization Wash Buffer I, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/permeabilization wash buffer i/product/R&D Systems
    Average 96 stars, based on 1 article reviews
    permeabilization wash buffer i - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier
    flow cytometry permeabilization wash buffer i 1x r d systems cat fc005  (R&D Systems)
    96
    R&D Systems flow cytometry permeabilization wash buffer i 1x r d systems cat fc005
    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow <t>cytometry</t> examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.
    Flow Cytometry Permeabilization Wash Buffer I 1x R D Systems Cat Fc005, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry permeabilization wash buffer i 1x r d systems cat fc005/product/R&D Systems
    Average 96 stars, based on 1 article reviews
    flow cytometry permeabilization wash buffer i 1x r d systems cat fc005 - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier
    flow cytometry permeabilization wash buffer  (R&D Systems)
    96
    R&D Systems flow cytometry permeabilization wash buffer
    Figure 1. Staining of cells for markers of pluripotency (A) Flow <t>cytometry</t> scatterplots from Exp.1-1-purified iPSCs. All plots have the green fluorescein isothiocyanate (FITC) channel on the x axis and side scatter on the y axis. The plot on the left shows an unstained control sample that was used to gate for positive Alexa 488 staining. The percentage of positive staining is shown in the top left corner of each plot. The middle and right plots show the TRA-1-60 Alexa 488 and SSEA-4 Alexa 488 stainings, respectively.
    Flow Cytometry Permeabilization Wash Buffer, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry permeabilization wash buffer/product/R&D Systems
    Average 96 stars, based on 1 article reviews
    flow cytometry permeabilization wash buffer - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier
    flow cytometry permeabilization wash buffer i 1x  (R&D Systems)
    96
    R&D Systems flow cytometry permeabilization wash buffer i 1x
    Figure 7. Emergence of βIII-tubulin (TUJ1, neuron) and glial fibrillary acidic protein (GFAP, astro- cyte) in neural progenitor cells derived from human induced pluripotent stem cells (hiPSCs-NPC) cultivated over the scaffolds after DIV 15 in free-specific growth factor conditions. (A) Schematic illustration of hiPSCs-NPC cultures. (B–D) TUJ1 marker plotted in flow <t>cytometry</t> graphs of hiPSC- NPC seeded on (B) poly (ε-caprolactone) scaffold (PCL), (C) poly (L-lactic acid) scaffold (PLLA), and (D) chitosan scaffold (CHI). (E–G) GFAP marker plotted in flow cytometry graphs of hiPSC-NPC seeded on (E) PCL, (F) PLLA, and (G) CHI. (H) Percentage of TUJ1 and GFAP in hiPSC-NPC on DIV 15. Bars represent standard errors of the mean. Illustration made on Biorender.com. * p < 0.05.
    Flow Cytometry Permeabilization Wash Buffer I 1x, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry permeabilization wash buffer i 1x/product/R&D Systems
    Average 96 stars, based on 1 article reviews
    flow cytometry permeabilization wash buffer i 1x - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier

    Image Search Results


    ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow cytometry examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Synthetic routes of the clickable PD-L1 inhibitors. ( B and C ) Flow cytometry examination of PD-L1 abundance and quantification of inhibition efficiency on the membrane surface of (B) wild-type 4T1 tumor cells and (C) wild-type B16-F10 tumor cells [median fluorescence intensity (MFI)]. The tumor cells were first incubated with Ac 4 ManAz (25 μM) for 3 days to label PD-L1 with azide groups and then treated with the clickable PD-L1 inhibitors for 24 hour. Last, the PD-L1 abundance on the membrane surface was examined by flow cytometry. ( D ) Schematic illustration of the positive correlation between the OEG linker length of the clickable PD-L1 inhibitors and their PD-L1 degradation efficacy. ( E and F ) Flow cytometry–determined PD-L1 abundance on the surface of (E) wild-type 4T1 tumor cells and (F) wild-type B16-F10 tumor cells without or without azide labeling. ( G ) A proposed mechanism for clickable PD-L1 inhibitor–mediated PD-L1 degradation via bioorthogonal click chemistry and metabolic glycan engineering, which is superior over the conventional inhibitors via physical binding. ( H ) Flow cytometry determined the membrane surface PD-L1 degradation profile of the clickable PD-L1 inhibitor in various human and murine tumor cell lines. The data are presented as means ± SD.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: Flow Cytometry, Inhibition, Membrane, Fluorescence, Incubation, Labeling, Binding Assay

    ( A ) Schematic illustration of IFN-γ–induced PD-L1 up-regulation on the surface of tumor cells in vitro. ( B to E ) Flow cytometry and Western blot examination of clickable PD-L1 inhibitor–mediated PD-L1 degradation on the surface of tumor cell membrane in vitro. [(B) and (C)] Flow cytometry detection of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (B) and B16-F10 (C) tumor cells. [(D) and (E)] Western blot analysis of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (D) and B16-F10 (E) tumor cells. (F) Representative CLSM images of PD-L1 abundance on the membrane surface of 4T1 tumor cells (scale bar = 20 μm). Na + ,K + -ATPase, Na + - and K + -dependent adenosine triphosphatase. ( G to J ) Flow cytometry–determined PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (G) and B16-F10 (H) tumor cells after treatment with 10 μM BMS-1 or D5B. Western blot analysis and semi-quantification of PD-L1 expression on the membrane surface of IFN-γ–pretreated 4T1 (I) and B16-F10 (J) tumor cells with or without azide labeling. ( K ) Representative flow cytometry plots and quantification of IFN-γ + CD8 + T cells. 1#, CD8 + T cells incubated with BMS-1–treated tumor cells; 2#, CD8 + T cells incubated with D5B-treated tumor cells; 3#, CD8 + T cells incubated with PBS. ( L ) Mechanism illustration for PD-L1 degradation increased proliferation of CD8 + T lymphocytes. The data were presented as the means ± SD. P values were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test [(D) and (E)], unpaired Student’s t test [(I) and (J)], or one-way ANOVA with Tukey’s multiple comparisons test (L). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Schematic illustration of IFN-γ–induced PD-L1 up-regulation on the surface of tumor cells in vitro. ( B to E ) Flow cytometry and Western blot examination of clickable PD-L1 inhibitor–mediated PD-L1 degradation on the surface of tumor cell membrane in vitro. [(B) and (C)] Flow cytometry detection of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (B) and B16-F10 (C) tumor cells. [(D) and (E)] Western blot analysis of PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (D) and B16-F10 (E) tumor cells. (F) Representative CLSM images of PD-L1 abundance on the membrane surface of 4T1 tumor cells (scale bar = 20 μm). Na + ,K + -ATPase, Na + - and K + -dependent adenosine triphosphatase. ( G to J ) Flow cytometry–determined PD-L1 abundance on the membrane surface of IFN-γ–pretreated 4T1 (G) and B16-F10 (H) tumor cells after treatment with 10 μM BMS-1 or D5B. Western blot analysis and semi-quantification of PD-L1 expression on the membrane surface of IFN-γ–pretreated 4T1 (I) and B16-F10 (J) tumor cells with or without azide labeling. ( K ) Representative flow cytometry plots and quantification of IFN-γ + CD8 + T cells. 1#, CD8 + T cells incubated with BMS-1–treated tumor cells; 2#, CD8 + T cells incubated with D5B-treated tumor cells; 3#, CD8 + T cells incubated with PBS. ( L ) Mechanism illustration for PD-L1 degradation increased proliferation of CD8 + T lymphocytes. The data were presented as the means ± SD. P values were determined by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test [(D) and (E)], unpaired Student’s t test [(I) and (J)], or one-way ANOVA with Tukey’s multiple comparisons test (L). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: In Vitro, Flow Cytometry, Western Blot, Membrane, Expressing, Labeling, Incubation

    ( A ) Acid-responsive mechanism illustration of the pH e -activatable PCP@D5B, pH i -activatable PDP@D5B, and pH-inactivated PBP@D5B nanoparticles for acid-triggered nanoparticle dissociation and activation of NIRF/MRI signals. ( B ) Dynamic light scattering (DLS)– and transmission electron microscopy (TEM)–determined particle size distribution and morphology change of the PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles at pH 7.4 and 6.5, respectively (scale bars, 100 nm). ( C ) Representative CLSM images of cell membrane [wheat germ agglutinin (WGA), green] and colocalization with PPa-labeled nanoparticles (red) at the predesignated time points. 4T1 tumor cells were incubated with PCP@D5B, PDP@D5B, or PBP@D5B nanoparticles for 5 min under different pH conditions (scale bars, 20 μm). ( D ) Schematic illustration for PCP@D5B-performed PD-L1 degradation on the surface of tumor cell membrane by specifically releasing D5B payload at the extracellular acidic microenvironment in vitro. ( E ) Flow cytometry analysis of PD-L1 abundance on the surface of 4T1 tumor cell membrane; inset number represents the MFI values. ( F ) The mechanism of pH-activated NIRF and MRI signals of PCPGd@D5B nanoparticles. ( G ) Representative T 1 maps of PCPGd@D5B nanoparticles determined at varied pH values. ( H ) The longitudinal relaxation rate ( r 1 ) versus Gd 3+ concentration determined at different pH values. ( I ) MRI of 4T1 tumor-bearing mice in vivo. The mice were intravenously injected with PBS or PCPGd@D5B nanoparticles at a Gd 3+ dose of 1.5 mg/kg and then imaged at the predetermined intervals (white circles represent the tumors). The data are presented as the means ± SD. h, hours.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Acid-responsive mechanism illustration of the pH e -activatable PCP@D5B, pH i -activatable PDP@D5B, and pH-inactivated PBP@D5B nanoparticles for acid-triggered nanoparticle dissociation and activation of NIRF/MRI signals. ( B ) Dynamic light scattering (DLS)– and transmission electron microscopy (TEM)–determined particle size distribution and morphology change of the PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles at pH 7.4 and 6.5, respectively (scale bars, 100 nm). ( C ) Representative CLSM images of cell membrane [wheat germ agglutinin (WGA), green] and colocalization with PPa-labeled nanoparticles (red) at the predesignated time points. 4T1 tumor cells were incubated with PCP@D5B, PDP@D5B, or PBP@D5B nanoparticles for 5 min under different pH conditions (scale bars, 20 μm). ( D ) Schematic illustration for PCP@D5B-performed PD-L1 degradation on the surface of tumor cell membrane by specifically releasing D5B payload at the extracellular acidic microenvironment in vitro. ( E ) Flow cytometry analysis of PD-L1 abundance on the surface of 4T1 tumor cell membrane; inset number represents the MFI values. ( F ) The mechanism of pH-activated NIRF and MRI signals of PCPGd@D5B nanoparticles. ( G ) Representative T 1 maps of PCPGd@D5B nanoparticles determined at varied pH values. ( H ) The longitudinal relaxation rate ( r 1 ) versus Gd 3+ concentration determined at different pH values. ( I ) MRI of 4T1 tumor-bearing mice in vivo. The mice were intravenously injected with PBS or PCPGd@D5B nanoparticles at a Gd 3+ dose of 1.5 mg/kg and then imaged at the predetermined intervals (white circles represent the tumors). The data are presented as the means ± SD. h, hours.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: Activation Assay, Transmission Assay, Electron Microscopy, Membrane, Labeling, Incubation, In Vitro, Flow Cytometry, Concentration Assay, In Vivo, Injection

    ( A ) Schematic illustration of pH-triggered extracellular delivery of D5B for PD-L1 degradation. ( B ) Representative IVIS fluorescence images of 4T1 tumor-bearing BALB/c mice in vivo. ( C ) Semiquantitative of PPa fluorescence intensity from (B) ( n = 3 mice). ( D ) High-performance liquid chromatography (HPLC)–determined pharmacokinetics of D5B-loaded PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles or free D5B ( n = 3 mice). ( E ) HPLC-determined D5B distribution in the tumor mass after intravenous injection ( n = 3 mice). ( F ) Experimental schedule for antitumor study in vivo. it, intratumoral; iv, intravenous; sc, subcutaneous. ( G and H ) Averaged tumor growth curves (G), and (H) animal survival curves of 4T1 tumor-bearing mice (n = 6 mice). ( I and J ) Immunohistochemical (IHC) (I) and flow cytometry (J) examination of PD-L1 abundance 3 days after treatment ( n = 3 mice; scale bars, 50 μm). ( K ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD3 + CD45 + ) ( n = 5 mice). ( L ) Flow cytometry examination of tumor-infiltrating IFN-γ + CD8 + T cells (n = 5 mice). ( M and N ) Tumor mass normalized number of tumor-infiltrating CD8 + (M) and IFN-γ + CD8 + T cells (N) ( n = 5 mice). ( O ) Enzyme-linked immunosorbent assay (ELISA) analysis of intratumoral IFN-γ cytokine secretion at 1, 3, and 7 days after treatment ( n = 3 mice). ( P ) IHC examination of PD-L1 abundance in the normal tissue 3 days after the treatment. ( Q ) Schematic description for tumor-specific delivery of D5B and PD-L1 inhibition with the pH e -activatable nanoparticles. All data are presented as the means ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test [(J) to (N)], repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(E), (G), and (O)], log-rank test (H), or unpaired Student’s t test (P). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Schematic illustration of pH-triggered extracellular delivery of D5B for PD-L1 degradation. ( B ) Representative IVIS fluorescence images of 4T1 tumor-bearing BALB/c mice in vivo. ( C ) Semiquantitative of PPa fluorescence intensity from (B) ( n = 3 mice). ( D ) High-performance liquid chromatography (HPLC)–determined pharmacokinetics of D5B-loaded PCP@D5B, PDP@D5B, and PBP@D5B nanoparticles or free D5B ( n = 3 mice). ( E ) HPLC-determined D5B distribution in the tumor mass after intravenous injection ( n = 3 mice). ( F ) Experimental schedule for antitumor study in vivo. it, intratumoral; iv, intravenous; sc, subcutaneous. ( G and H ) Averaged tumor growth curves (G), and (H) animal survival curves of 4T1 tumor-bearing mice (n = 6 mice). ( I and J ) Immunohistochemical (IHC) (I) and flow cytometry (J) examination of PD-L1 abundance 3 days after treatment ( n = 3 mice; scale bars, 50 μm). ( K ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD3 + CD45 + ) ( n = 5 mice). ( L ) Flow cytometry examination of tumor-infiltrating IFN-γ + CD8 + T cells (n = 5 mice). ( M and N ) Tumor mass normalized number of tumor-infiltrating CD8 + (M) and IFN-γ + CD8 + T cells (N) ( n = 5 mice). ( O ) Enzyme-linked immunosorbent assay (ELISA) analysis of intratumoral IFN-γ cytokine secretion at 1, 3, and 7 days after treatment ( n = 3 mice). ( P ) IHC examination of PD-L1 abundance in the normal tissue 3 days after the treatment. ( Q ) Schematic description for tumor-specific delivery of D5B and PD-L1 inhibition with the pH e -activatable nanoparticles. All data are presented as the means ± SD. P values were determined by one-way ANOVA with Tukey’s post hoc test [(J) to (N)], repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(E), (G), and (O)], log-rank test (H), or unpaired Student’s t test (P). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not significant.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: Fluorescence, In Vivo, High Performance Liquid Chromatography, Injection, Immunohistochemical staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Inhibition

    ( A ) Treatment schedule of in 4T1 tumor model in vivo. ( B ) Individual 4T1 tumor growth curves [complete regression (CR)] ( n = 6 mice). ( C ) Survival rates of 4T1 tumor-bearing mice. ( D ) Flow cytometry analysis of CD86 + CD80 + DCs ( n = 3 mice). ( E ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells, and ( F ) IFN-γ + CD8 + T cells ( n = 5 mice). ( G to I ) Absolute numbers of tumor-infiltrating CD3 + (G), CD8 + (H), and IFN-γ + CD8 + (I) T cells after the indicated treatments ( n = 5 mice). ( J ) M2/M1 ratio after treatment ( n = 5 mice). ( K ) Flow cytometry–determined PD-L1 abundance on the surface of tumor cells membrane ( n = 5 mice). ( L ) Treatment schedule of 4T1 abscopal tumor model (T1 and T2 represents the primary and abscopal tumors, respectively). ( M ) Averaged tumor growth curves ( n = 6 mice), and ( N ) Survival rates of the mice ( n = 6 mice). ( O ) Flow cytometry–determined tumor-infiltrating CD8 + T cells. ( P ) Flow cytometry analysis of T EM cells (CD62L − CD44 + ) in the spleens of 4T1 tumor-bearing mice ( n = 5 mice). ( Q ) Hematoxylin and eosin staining and quantification of metastatic tumor lesions in the lung ( n = 6 mice; scale bars, 2.5 mm). ( R ) Mechanism illustration for combinatory therapy–elicited antitumor immunity and immunological memory to suppress abscopal tumor and lung metastases. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(B) and (M)], log-rank test [(C) and (N)], or one-way ANOVA with Tukey’s post hoc test [(D) to (K) and (O) to (Q)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Treatment schedule of in 4T1 tumor model in vivo. ( B ) Individual 4T1 tumor growth curves [complete regression (CR)] ( n = 6 mice). ( C ) Survival rates of 4T1 tumor-bearing mice. ( D ) Flow cytometry analysis of CD86 + CD80 + DCs ( n = 3 mice). ( E ) Flow cytometry examination of tumor-infiltrating CD8 + and CD4 + T cells, and ( F ) IFN-γ + CD8 + T cells ( n = 5 mice). ( G to I ) Absolute numbers of tumor-infiltrating CD3 + (G), CD8 + (H), and IFN-γ + CD8 + (I) T cells after the indicated treatments ( n = 5 mice). ( J ) M2/M1 ratio after treatment ( n = 5 mice). ( K ) Flow cytometry–determined PD-L1 abundance on the surface of tumor cells membrane ( n = 5 mice). ( L ) Treatment schedule of 4T1 abscopal tumor model (T1 and T2 represents the primary and abscopal tumors, respectively). ( M ) Averaged tumor growth curves ( n = 6 mice), and ( N ) Survival rates of the mice ( n = 6 mice). ( O ) Flow cytometry–determined tumor-infiltrating CD8 + T cells. ( P ) Flow cytometry analysis of T EM cells (CD62L − CD44 + ) in the spleens of 4T1 tumor-bearing mice ( n = 5 mice). ( Q ) Hematoxylin and eosin staining and quantification of metastatic tumor lesions in the lung ( n = 6 mice; scale bars, 2.5 mm). ( R ) Mechanism illustration for combinatory therapy–elicited antitumor immunity and immunological memory to suppress abscopal tumor and lung metastases. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test [(B) and (M)], log-rank test [(C) and (N)], or one-way ANOVA with Tukey’s post hoc test [(D) to (K) and (O) to (Q)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: In Vivo, Flow Cytometry, Membrane, Staining

    ( A ) Treatment schedule for the antitumor study in B16-F10 tumor-bearing mice in vivo. ( B ) The averaged and individual B16-F10 tumor growth curves, and ( C ) survival curves of B16-F10 tumor-bearing mice monitored during the therapy period (CR represents the fractions of complete tumor regression at the end of antitumor study, n = 6 or 7 mice). ( D and E ) Representative flow cytometry plots (D), and quantification data of CD86 + CD80 + DCs (E) ( n = 3 mice). ( F and G ) Flow cytometry–determined fractions of (F) M2-phenotype (CD11b + CD206 + ) and (G) M1-phenotype (CD11b + CD80 + ) TAMs ( n = 5 mice). ( H ) The M2/M1 ratio of TAMs. ( I and J ) The MFIs of PD-L1 + TAMs (CD11b + CD80 + ) (I), and PD-L1 + CD45 − tumor cells (J) after treatment. ( K ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs in the tumor sections (scale bars, 40 μm). ( L and M ) Representative flow cytometry plots (L) and quantification (M) of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD45 + CD3 + ) ( n = 5 mice). ( N ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs, and CD8 + T cells in the tumor sections (scale bars, 40 μm). ( O ) Schematic illustration of the clickable PD-L1 inhibitor mitigated the acquired immune evasion. RT induces ITM by up-regulating PD-L1 and recruiting M2-type TAMs, which was reversed with the clickable PD-L1 inhibitor through degrading PD-L1 on the surface of tumor cell membrane and repolarizing M2-type TAMs to M1 type. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test (B), log-rank test (C), or one-way ANOVA with Tukey’s post hoc test [(E) to (J) and (M)], * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Journal: Science Advances

    Article Title: Tumor-specific delivery of clickable inhibitor for PD-L1 degradation and mitigating resistance of radioimmunotherapy

    doi: 10.1126/sciadv.adq3940

    Figure Lengend Snippet: ( A ) Treatment schedule for the antitumor study in B16-F10 tumor-bearing mice in vivo. ( B ) The averaged and individual B16-F10 tumor growth curves, and ( C ) survival curves of B16-F10 tumor-bearing mice monitored during the therapy period (CR represents the fractions of complete tumor regression at the end of antitumor study, n = 6 or 7 mice). ( D and E ) Representative flow cytometry plots (D), and quantification data of CD86 + CD80 + DCs (E) ( n = 3 mice). ( F and G ) Flow cytometry–determined fractions of (F) M2-phenotype (CD11b + CD206 + ) and (G) M1-phenotype (CD11b + CD80 + ) TAMs ( n = 5 mice). ( H ) The M2/M1 ratio of TAMs. ( I and J ) The MFIs of PD-L1 + TAMs (CD11b + CD80 + ) (I), and PD-L1 + CD45 − tumor cells (J) after treatment. ( K ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs in the tumor sections (scale bars, 40 μm). ( L and M ) Representative flow cytometry plots (L) and quantification (M) of tumor-infiltrating CD8 + and CD4 + T cells (gated on CD45 + CD3 + ) ( n = 5 mice). ( N ) Immunofluorescence staining and semi-quantitation of PD-L1 + TAMs, and CD8 + T cells in the tumor sections (scale bars, 40 μm). ( O ) Schematic illustration of the clickable PD-L1 inhibitor mitigated the acquired immune evasion. RT induces ITM by up-regulating PD-L1 and recruiting M2-type TAMs, which was reversed with the clickable PD-L1 inhibitor through degrading PD-L1 on the surface of tumor cell membrane and repolarizing M2-type TAMs to M1 type. The data are presented as the means ± SD. P values were determined by repeated-measures two-way ANOVA with Tukey’s multiple comparisons test (B), log-rank test (C), or one-way ANOVA with Tukey’s post hoc test [(E) to (J) and (M)], * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

    Article Snippet: To stain intracellular proteins, the cell suspension was fixed and permeabilized with the commercial buffer Flow Cytometry Permeabilization/Wash Buffer I (R&D Systems), followed by intracellular staining with anti–IFN-γ–FITC.

    Techniques: In Vivo, Flow Cytometry, Immunofluorescence, Staining, Quantitation Assay, Membrane

    Figure 1. Staining of cells for markers of pluripotency (A) Flow cytometry scatterplots from Exp.1-1-purified iPSCs. All plots have the green fluorescein isothiocyanate (FITC) channel on the x axis and side scatter on the y axis. The plot on the left shows an unstained control sample that was used to gate for positive Alexa 488 staining. The percentage of positive staining is shown in the top left corner of each plot. The middle and right plots show the TRA-1-60 Alexa 488 and SSEA-4 Alexa 488 stainings, respectively.

    Journal: Cell reports methods

    Article Title: Reliable multiplex generation of pooled induced pluripotent stem cells.

    doi: 10.1016/j.crmeth.2023.100570

    Figure Lengend Snippet: Figure 1. Staining of cells for markers of pluripotency (A) Flow cytometry scatterplots from Exp.1-1-purified iPSCs. All plots have the green fluorescein isothiocyanate (FITC) channel on the x axis and side scatter on the y axis. The plot on the left shows an unstained control sample that was used to gate for positive Alexa 488 staining. The percentage of positive staining is shown in the top left corner of each plot. The middle and right plots show the TRA-1-60 Alexa 488 and SSEA-4 Alexa 488 stainings, respectively.

    Article Snippet: The cells were resuspended with 100ul of 4% paraformaldehyde (Electron Microscopy Sciences 15710) in DPBS and incubated for 20 min at 4 C. The cells were then washed twice with Flow Cytometry Permeabilization/Wash Buffer (R&D Systems FC005) before being pelleted and resuspended in 50ul of permeabilization wash buffer with 1:50 of each antibody for 1 h at 4 C. The antibodies used were SOX2 and POU5F1P5 (OCT4) both conjugated with Alexa Fluor 647 (Abcam ab196637 and ab300092 respectively), as well as Mouse IgG2A kappa (BioLegend 400234).

    Techniques: Staining, Flow Cytometry, Control

    Figure 5. Differentiation of pooled iPSCs into brain organoids (A) Bright-field images of intact 2-month-old brain organoids differentiated from pooled iPSCs. Individual organoids display variability in morphology. (B) Flow cytometry scatterplots from 2-month-old brain dissociated organoids. All plots have the red Alexa Fluor 647 channel on the x axis and side scatter on the y axis. The first plot shows a control sample stained with mouse IgG2a kappa, which was used to create the black gate for positive Alexa 647 staining. The percentage positive is shown within the gate of each plot. The second plot shows TUBB3 (TuJ1) staining, the third plot shows NES (Nestin) staining, and the fourth plot shows P2RY12 staining. (C) Immunohistochemistry labeling of an intact 2-month-old brain organoid. All images were taken at 253 magnification. TuJ1 was labeled with an Alexa 488-conjugated antibody, shown in the first image. Nestin was labeled with an Alexa 647-conjugated antibody, shown in the second image. The third image is an overlay of TuJ1 and Nestin labeling. The fourth image is an overlay of TuJ1, Nestin, and DAPI staining in blue.

    Journal: Cell reports methods

    Article Title: Reliable multiplex generation of pooled induced pluripotent stem cells.

    doi: 10.1016/j.crmeth.2023.100570

    Figure Lengend Snippet: Figure 5. Differentiation of pooled iPSCs into brain organoids (A) Bright-field images of intact 2-month-old brain organoids differentiated from pooled iPSCs. Individual organoids display variability in morphology. (B) Flow cytometry scatterplots from 2-month-old brain dissociated organoids. All plots have the red Alexa Fluor 647 channel on the x axis and side scatter on the y axis. The first plot shows a control sample stained with mouse IgG2a kappa, which was used to create the black gate for positive Alexa 647 staining. The percentage positive is shown within the gate of each plot. The second plot shows TUBB3 (TuJ1) staining, the third plot shows NES (Nestin) staining, and the fourth plot shows P2RY12 staining. (C) Immunohistochemistry labeling of an intact 2-month-old brain organoid. All images were taken at 253 magnification. TuJ1 was labeled with an Alexa 488-conjugated antibody, shown in the first image. Nestin was labeled with an Alexa 647-conjugated antibody, shown in the second image. The third image is an overlay of TuJ1 and Nestin labeling. The fourth image is an overlay of TuJ1, Nestin, and DAPI staining in blue.

    Article Snippet: The cells were resuspended with 100ul of 4% paraformaldehyde (Electron Microscopy Sciences 15710) in DPBS and incubated for 20 min at 4 C. The cells were then washed twice with Flow Cytometry Permeabilization/Wash Buffer (R&D Systems FC005) before being pelleted and resuspended in 50ul of permeabilization wash buffer with 1:50 of each antibody for 1 h at 4 C. The antibodies used were SOX2 and POU5F1P5 (OCT4) both conjugated with Alexa Fluor 647 (Abcam ab196637 and ab300092 respectively), as well as Mouse IgG2A kappa (BioLegend 400234).

    Techniques: Flow Cytometry, Control, Staining, Immunohistochemistry, Labeling

    Figure 7. Emergence of βIII-tubulin (TUJ1, neuron) and glial fibrillary acidic protein (GFAP, astro- cyte) in neural progenitor cells derived from human induced pluripotent stem cells (hiPSCs-NPC) cultivated over the scaffolds after DIV 15 in free-specific growth factor conditions. (A) Schematic illustration of hiPSCs-NPC cultures. (B–D) TUJ1 marker plotted in flow cytometry graphs of hiPSC- NPC seeded on (B) poly (ε-caprolactone) scaffold (PCL), (C) poly (L-lactic acid) scaffold (PLLA), and (D) chitosan scaffold (CHI). (E–G) GFAP marker plotted in flow cytometry graphs of hiPSC-NPC seeded on (E) PCL, (F) PLLA, and (G) CHI. (H) Percentage of TUJ1 and GFAP in hiPSC-NPC on DIV 15. Bars represent standard errors of the mean. Illustration made on Biorender.com. * p < 0.05.

    Journal: International journal of molecular sciences

    Article Title: The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

    doi: 10.3390/ijms241713642

    Figure Lengend Snippet: Figure 7. Emergence of βIII-tubulin (TUJ1, neuron) and glial fibrillary acidic protein (GFAP, astro- cyte) in neural progenitor cells derived from human induced pluripotent stem cells (hiPSCs-NPC) cultivated over the scaffolds after DIV 15 in free-specific growth factor conditions. (A) Schematic illustration of hiPSCs-NPC cultures. (B–D) TUJ1 marker plotted in flow cytometry graphs of hiPSC- NPC seeded on (B) poly (ε-caprolactone) scaffold (PCL), (C) poly (L-lactic acid) scaffold (PLLA), and (D) chitosan scaffold (CHI). (E–G) GFAP marker plotted in flow cytometry graphs of hiPSC-NPC seeded on (E) PCL, (F) PLLA, and (G) CHI. (H) Percentage of TUJ1 and GFAP in hiPSC-NPC on DIV 15. Bars represent standard errors of the mean. Illustration made on Biorender.com. * p < 0.05.

    Article Snippet: 2023, 24, 13642 20 of 25 USA) and incubated for 30 min at room temperature, then washed and resuspended in flow cytometry permeabilization/wash buffer I (1X) (FC005, R&D Systems, Minneapolis, MN, USA).

    Techniques: Derivative Assay, Marker, Cytometry