rabbit anti mouse hmgb1 antibody  (Abcam)

Journal: Journal of Nanobiotechnology

Article Title: Multifunctional nanoagonist enhances photodynamic therapy-driven in situ cancer vaccination by inhibiting tumor thrombosis

doi: 10.1186/s12951-025-03843-8

Figure Lengend Snippet: In vitro evaluation of cDVPMA-induced ICD effects. ( A ) Representative CLSM images of CRT exposure on 4T1 cells following different treatments. Scale bar = 20 μm. ( B ) Representative CLSM images of HMGB1 release from 4T1 cells following different treatments. Scale bar = 20 μm. ( C ) Quantitative analysis of CRT exposure on 4T1 cells following different treatments. ( D ) Quantitative analysis of HMGB1 release from 4T1 cells following different treatments. ELISA analysis of ( E ) HMGB1 and ( F ) ATP release from 4T1 cells following different treatments. ( G ) Western blot analysis and quantification of ( H ) p-STING activation, ( I ) p-TBK1 activation, ( J ) p-IRF3 activation after various treatments. ( K ) Scheme illustration of the transwell co-culture system. Concentrations of ( L ) IFN-β and ( M ) TNF-α in the supernatants of different treatment groups. ( N ) Quantitative analysis and ( O ) representative flow cytometry plots of the proportion of mature DCs after co-incubation with 4T1 cell supernatants pre-stimulated with different formulations. FL., fluorescence intensity. c, free 2′3′-cGAMP. All data are represented as mean ± SD ( n = 3). ns, no significance. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Singlet Oxygen Sensor Green (SOSG) fluorescent probe, dichlorodihydrofluorescein diacetate (DCFH-DA), cytokine detection kits (IFN-β, IFN-γ, IL-6, TNF-α, IL-1β, TGF-β1), ATP detection kit, 4′,6-diamidino-2-phenylindole (DAPI), protease inhibitors, Alexa Fluor 555-labeled donkey anti-rabbit IgG (H + L), rabbit anti-mouse β -actin monoclonal antibody, HRP-labeled goat anti-rabbit IgG (H + L), HMGB1 detection kit, GM-CSF, and IL-4 were all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. Rabbit anti-mouse calreticulin polyclonal antibody and rabbit anti-mouse HMGB1 polyclonal antibody were purchased from Proteintech Group, Inc. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and trypsin were obtained from Thermo Fisher Scientific Inc. Phospho-STING (Ser365) (D8F4W) rabbit monoclonal antibody (#72971), and Phospho-IRF-3 (Ser396) (4D4G) rabbit monoclonal antibody (#4947) were purchased from Cell Signaling Technology, Inc. Anti-NAK/TBK1 (phospho S172) antibody [EPR2867(2)] was obtained from Abcam.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Co-Culture Assay, Flow Cytometry, Incubation, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Bacteria-driven nanosonosensitizer delivery system for enhanced breast cancer treatment through sonodynamic therapy-induced immunogenic cell death

doi: 10.1186/s12951-024-02437-0

Figure Lengend Snippet: The efficiency of ICD induction in vitro. ( A - B ) CRT exposure level on the tumor cell surface and its quantification: Green: CRT; Blue: DAPI staining. ( C - D ) Intracellular HMGB1 secretion level and its quantification in the tumor cells: Green: HMGB1; Blue: DAPI staining. (* p < 0.05, ** p < 0.01, **** p < 0.0001)

Article Snippet: The cells were first incubated with rabbit anti-mouse CRT primary antibody and rabbit anti-mouse HMGB1 primary antibody (Beyotime, Shanghai, China) followed by incubation with Alexa Fluor 488-coupled sheep anti-rabbit secondary antibody.

Techniques: In Vitro, Staining

Journal: Journal of Nanobiotechnology

Article Title: Bacteria-driven nanosonosensitizer delivery system for enhanced breast cancer treatment through sonodynamic therapy-induced immunogenic cell death

doi: 10.1186/s12951-024-02437-0

Figure Lengend Snippet: In vivo anti-tumor metastasis and ICD measurement. ( A ) Number of pulmonary metastases (a: Control; b: US; c: HPNDs@EcN; d, HPNDs@EcN + US). ( B ) Immunohistochemistry of CRT and HMGB1 in tumor tissue after various treatments. ( C - D ) Flow quantification of mature dendritic cells in tumor tissue after various treatments. ( E , F ) Flow quantification of infiltrated CD8 + T cells in tumor tissue after various treatments. (** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The cells were first incubated with rabbit anti-mouse CRT primary antibody and rabbit anti-mouse HMGB1 primary antibody (Beyotime, Shanghai, China) followed by incubation with Alexa Fluor 488-coupled sheep anti-rabbit secondary antibody.

Techniques: In Vivo, Control, Immunohistochemistry

Journal: Advanced Science

Article Title: Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti‐Tumor Immunity

doi: 10.1002/advs.202305089

Figure Lengend Snippet: The tumor‐released HMGB1‐gDNA complex activates cGAS‐STING and NF‐kB pathways, and the tumor‐released oxLDL induces inflammasome activation in DCs. a) Relative abundance of gDNA and mtDNA in the cGAS immunoprecipitates of DC2.4 cells that were cocultured with either the Scramble‐ or Arf1‐ablated CT26 cells ( n = 3). b) Relative abundance of cytosolic gDNA and mtDNA in DC2.4 cells that were cocultured with either the Scramble‐ or Arf1‐ablated CT26 cells ( n = 3). c) DC2.4 cells were cocultured with either the Scramble‐ or Arf1‐ablated human DLD1 colorectal tumor cells, the cytosolic human gDNA, and mtDNA in DCs were examined by RT‐qPCR ( n = 3). d) Relative abundance of gDNA in the HMGB1 immunoprecipitates of the collected culture medium from either the Scramble‐ or Arf1‐ablated CT26 cells ( n = 3). e) Immunoblotting analysis of the indicated proteins in DC2.4 cells that were cocultured with either the Scramble (Sc)‐ or Arf1‐ablated CT26 cells in presence or absence of the indicated concentrations of anti‐HMGB1 antibody. f) The secreted IFNβ was measured from DC2.4 cells that were treated as in (e) ( n = 2‐4). g) Immunoblotting analysis of the indicated proteins in DC2.4 cells that were treated as in (e). h) The secreted IL‐1β was measured from DC2.4 cells that were treated as in (e) ( n = 2‐3). i) The secreted oxLDL was measured from the medium of either the Scramble‐ or Arf1‐ablated CT26 cells ( n = 4). j) Immunoblotting analysis of pro‐IL‐1β and bioactive IL‐1β in DC 2.4 cells that were treated with or without oxLDL and LDL. k, l) Immunofluorescence staining of ASC (k) and quantification of ASC specks (l) in DC2.4 cells that were treated with or without exogenous oxLDL. Scale bars, 25 µm. m) Immunofluorescence staining of caspase‐1 and oxLDL in DC2.4 cells that were treated with or without exogenous Dil‐OxLDL. Scale bars, 25 µm. Each point represents the percentage of ASC specks per macroscopic field (l). In all of the panels, data are presented as means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s., not significant. Student's t ‐test.

Article Snippet: The tumor supernatants or the collected cellular lysis was prepared to perform immunoprecipitation and incubated with rabbit anti‐mouse HMGB1 antibody (Cat# MA5‐31967, ThermoFisher, 1:50) or rabbit anti‐mouse cGAS antibody (Cat# 31659, Cell Signaling Technology, 1:50) overnight at 4 °C.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet: The expression of PLGA/PEI-GPC3 and PLGA/PEI-HMGB1 vaccine (A and B) The protein expression of HMGB1 or GPC3 was evaluated by flow cytometry in pHMGB1-transfected or pGPC3-transfected 293T cells. (C and D) Statistical analysis of the proportions of the positive cells in A and B. (E and F) The mice were intramuscularly administered PLGA/PEI-GPC3 and PLGA/PEI-HMGB1 vaccines. The injected muscular tissues were collected at 72 h after immunization. In vivo protein expression of HMGB1 and GPC3 was determined by western blot assay in muscular tissues. Data are from one representative experiment of three performed and presented as the mean ± SD, ∗∗∗∗p < 0.001.

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Expressing, Flow Cytometry, Transfection, Vaccines, Injection, In Vivo, Western Blot

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet: Anti-tumor effect of PLGA/PEI-HMGB1/GPC3 vaccine in the subcutaneous Hepa1-6-hGPC3 tumor model (A) The frequency of hGPC3-positive cells was assessed by flow cytometry in Hepa1-6 cells infected with hGPC3-lentivirus. C57BL/6 mice were s.c. implanted with 5×10 6 Hepa1-6-hGPC3 cells on the right flank. One week following inoculation, the mice were randomly sub-grouped and respectively vaccinated with PLGA/PEI-Vector, PLGA/PEI-HMGB1, PLGA/PEI-GPC3, or PLGA/PEI-HMGB1/GPC3 vaccines. (B) Tumor growth was recorded once a week from day 7 to day 42. (C) Tumor weights. (D) Tumor inhibition rate. (E and F) Immune cell subsets, including CD3 + T, CD8 + T, CD4 + T, DCs, NK, Mφ, and MDSCs, were quantified by flow cytometry in splenocytes and TILs. (G and H) The frequency of CD8 + CD11C + or CD103 + CD11C + in the splenocytes and TILs was detected by flow cytometry. Data are from one representative experiment of three performed and presented as the mean ± SD, ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Flow Cytometry, Infection, Plasmid Preparation, Vaccines, Inhibition

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet: CTL effect induced by PLGA/PEI-HMGB1/GPC3 co-immunization The lymphocytes obtained from the spleens were restimulated with GPC3 protein (10 μg/mL) ex vivo . (A and B) The proliferation ability of CD8 + T was assessed by EdU assay. (C and D) The frequencies of IFN-γ + CD8 + T, IL-2 + CD8 + T, or TNF-α + CD8 + T cells were analyzed by flow cytometry in GPC3 protein stimulated lymphocytes following incubation with 500 ng/mL Ionomycin, 50 ng/mL PMA, and 1× Brefeldin A for 5h. (E) The number of IFN-γ secreting CD8 + T cells was assessed by ELISPOT assay. (F) A co-culture experiment evaluated the antigen clearance effect. Data are from one representative experiment of three performed and presented as the mean ± SD, ∗∗p < 0.01.

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Ex Vivo, EdU Assay, Flow Cytometry, Incubation, Enzyme-linked Immunospot, Co-Culture Assay

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet: The anti-tumor effect induced by PLGA/PEI-HMGB1/GPC3 vaccine was CD8 + T cell dependent For the CD8 + T cell depletion assay, 0.5 mg CD8α antibody was administered two days before the tumor establishment and repeated on day 5 and day 12 after the first dose of vaccine. (A and B) The ratio of CD8 + T or CD8 + CD11C + subsets from the splenocytes or TILs was assessed by flow cytometry. (C) CD8 + T infiltration was detected by IHC assay (×200 magnification). 5×10 6 Hepa1-6-GPC3 cells were s.c. implanted in naive mice or PLGA/PEI-HMGB1-GPC3 vaccine recipients. (D) Tumor growth curves in vivo . The mice were euthanized on day 42, and tumor weight. (E) as well as tumor inhibition rate. (F) were measured. Data are from one representative experiment of three performed and presented as the mean ± SD, ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Depletion Assay, Flow Cytometry, In Vivo, Inhibition

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet: The induction of long-lasting anti-tumor effects by PLGA/PEI-HMGB1-GPC3 vaccine Mice that received PLGA/PEI-HMGB1-GPC3 vaccine were rechallenged with Hepa1-6-hGPC3 cells on the left flank accompanied by naive mice as the control group. (A) Growth curve of individual mice over time in 70 days. (B) The percentage survival of the control group and PLGA/PEI-HMGB1-GPC3 immunized group. (C) Representative images of flow cytometry and statistical analysis of CD8 + T cell cells labeled by CD44 and CD62L in the splenocytes from various vaccine immunization groups. (D–F) The frequencies of Naive: CD44 - /CD62L high , Central memory: CD44 + /CD62L high , Effector memory: CD44 + /CD62L low CD8 + CD11C + or CD103 + CD11C + in and TILs were analyzed statistically. Data are from one representative experiment of three performed and presented as the mean ± SD, ∗∗p < 0.01.

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Control, Flow Cytometry, Labeling

Journal: iScience

Article Title: HMGB1/GPC3 dual targeting vaccine induces dendritic cells-mediated CD8 + T cell immune response and elicits potential therapeutic effect in hepatocellular carcinoma

doi: 10.1016/j.isci.2023.106143

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse/human HMGB1 , Beyotime , Cat#AG2167.

Techniques: Virus, Recombinant, Enzyme-linked Immunospot, Software

Journal: Frontiers in Immunology

Article Title: Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation

doi: 10.3389/fimmu.2023.1012799

Figure Lengend Snippet: Unilateral histotripsy tumor ablation induces antigen-specific abscopal inhibition of distant, untreated tumors and local immunogenic cell death. (A) C57BL/6 mice were inoculated with bilateral flank B16F10 tumors (“HT abscopal concordant”) or unilateral B16F10 flank tumors and contralateral Hepa1-6 flank tumors (“HT abscopal discordant”), and sham (“control”) or histotripsy ablation encompassing ~80-90% of unilateral B16F10 flank tumors (in control and abscopal concordant groups) or unilateral Hepa1-6 flank tumors (in the abscopal discordant group) was performed on day 10. In contrast to sham ablation controls, mice treated with unilateral partial histotripsy ablation demonstrated immediate local growth arrest of treated B16F10 tumors (“HT treated”) and immediate abscopal growth inhibition of distant untreated B16F10 tumors (“HT abscopal concordant”) but not distant untreated B16F10 tumors after contralateral Hepa1-6 tumor ablation (“HT abscopal discordant”). (B) In mice bearing bilateral flank Hepa1-6 tumors or unilateral Hepa1-6 flank tumors and contralateral B16F10 flank tumors, unilateral partial histotripsy ablation of Hepa1-6 tumors (in control and HT abscopal concordant groups) or B16F10 tumors (in the HT abscopal discordant group) demonstrated immediate growth arrest and regression of treated Hepa1-6 tumors (“HT treated”) and distant untreated Hepa1-6 tumors (“HT abscopal concordant”), but not of distant untreated Hepa1-6 tumors after contralateral B16F10 tumor ablation (“HT abscopal discordant”). (C) Multicolor immunofluorescence analysis of bilateral B16F10 tumors performed 2 days after unilateral sham or partial histotripsy ablation demonstrated homogeneous intranuclear staining of HMGB1 in control tumors. In contrast, significant loss of intranuclear HMGB1 staining was observed within the ablation zone of histotripsy-treated tumors; intranuclear HMGB1 was retained outside of the ablation zone. (D) RNASeq of CD45- tumor cells performed on day 13 revealed marked differences in transcriptional activity between control tumors (blue) and histotripsy-treated (“HT-Tx”) tumors (red) as evidenced by principal component analysis. (E) qRT-PCR of tumors performed on day 13 revealed an approximately 20-fold increase in TNFα mRNA in histotripsy-treated tumors (red) compared with control tumors (blue). (F) Multicolor immunohistochemistry 1 day after sham or histotripsy ablation revealed no measurable expression of the necroptosis markers pRIPK3 and pMLKL in control tumors; in contrast, profound co-localized expression of pRIPK3 and pMLKL was seen along the periphery of ablated zones in histotripsy-treated tumors. (G) Serial quantitation of pRIPK3 fluorescence intensity in control (“C”) and histotripsy-treated (“HT-Tx”) tumors over various time points demonstrated rapid and transient upregulation of necroptosis-associated phosphorylated protein levels following histotripsy ablation. [ (A, B): n=7-9 mice per group; *=p<0.05 compared with control tumors; †=p<0.05 compared with HT abscopal concordant tumors. (C-G): n=3-4 mice per group; *=p<0.05 compared with control day 1 tumors; **=p < 0.01 compared with control day 1 tumors; ***=p < 0.0001 compared with control day 1 tumors].

Article Snippet: Samples were then incubated with rabbit anti-mouse HMGB1 antibody directly conjugated to Alexa 488 (Abcam) for 1 hour at 37°C.

Techniques: Inhibition, Immunofluorescence, Staining, Activity Assay, Quantitative RT-PCR, Immunohistochemistry, Expressing, Quantitation Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation

doi: 10.3389/fimmu.2023.1012799

Figure Lengend Snippet: Early cellular stress and immunogenic cell death is observed in treated tumors but not distant untreated tumors following histotripsy. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. (A) Multicolor immunofluorescence microscopy analysis of control, histotripsy-treated, and histotripsy-abscopal tumors on days 1, 3 or 10-12 after unilateral sham or partial histotripsy ablation revealed no significant release of intranuclear HMGB1 in control tumors at any time point. Histotripsy-treated tumors exhibited significant and immediate loss of nuclear HMGB1 staining that was highest within the ablation zone and the junction of ablated and peripheral non-ablated zones at early time points. At later time points, progressive loss of nuclear HMGB1 was observed within peripheral non-ablated zones of histotripsy-treated tumors. Similarly, whereas contralateral histotripsy-abscopal tumors demonstrated no HMGB1 translocation at early time points, progressive loss of intranuclear HMGB1 staining was observed at later time points. (B) RNASeq of CD45- tumor cells from sham-treated control tumors (blue) and histotripsy-abscopal (“HT-Abs”) tumors (red) performed 3 days after unilateral partial histotripsy revealed no substantial differences in overall mRNA transcriptional activity as evidenced by principal component analysis. (C) RNASeq of CD45- tumor cells 3 days after sham or unilateral histotripsy ablation demonstrated upregulated transcription of genes associated with necroptosis, ER stress, cellular response to LPS, TNFα signaling and inflammatory response in histotripsy-treated tumors (“HT-Tx”) as compared to control and histotripsy-abscopal (“HT-Abs”) tumors. (n=3-5 mice per experimental group).

Article Snippet: Samples were then incubated with rabbit anti-mouse HMGB1 antibody directly conjugated to Alexa 488 (Abcam) for 1 hour at 37°C.

Techniques: Immunofluorescence, Microscopy, Staining, Translocation Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation

doi: 10.3389/fimmu.2023.1012799

Figure Lengend Snippet: Late intratumoral CD8+ cell infiltration co-localizes with immunogenic and ferroptotic cancer cell death following histotripsy. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. (A) Multicolor immunofluorescence performed on day 7 after sham or histotripsy ablation revealed minimal CD8+ cell infiltration and minimal extranuclear translocation of HMGB1 among cancer cells in control tumors. CD8+ cell infiltration was co-localized with loss of intranuclear HMGB1 staining in histotripsy-treated tumors (B) and in histotripsy-abscopal tumors (C) . (D) Significantly higher percentages of cancer cells located within 50 μm of a CD8+ cell exhibited loss of nuclear HMGB1 in histotripsy-treated and histotripsy-abscopal tumors as compared with controls. (E) Multicolor immunofluorescence performed on days 3, 5 and 7 after sham or histotripsy ablation revealed spatial co-localization of CD8+ cell infiltration with cancer cell accumulation of 4-HNE, a byproduct of ferroptosis, in histotripsy-treated tumors but not in control tumors. (F) Strong co-localization was observed between CD8+ cell infiltration and 4-HNE on day 7 in histotripsy-treated and histotripsy-abscopal tumors, but not in control tumors. (G) Gradual accumulation of 4-HNE staining intensity was observed in histotripsy-treated (“HT Tx”) and histotripsy-abscopal (“HT Abs”) tumor cells. (H) A linear relationship was observed between number of CD8+ cells present (x axis) and intensity of 4-HNE expression within 50 μm (y-axis) in histotripsy-treated tumors but not in control tumors. (I, J) Mice bearing B16F10 flank tumors were treated with sham or histotripsy tumor ablation on day 10, and tumor-draining lymph nodes were harvested on day 15. CD8+ T cells derived from tumor-draining lymph nodes were co-cultured with B16F10 melanoma cells in vitro . Significant accumulation of 4-HNE was observed within B16F10 melanoma cells co-cultured with CD8+ T cells derived from lymph nodes draining histotripsy-treated but not sham-treated tumors. (n=3-6 mice per group; *=p < 0.05 compared with control day 1 tumors; ***=p < 0.001 compared with control day 1 tumors).

Article Snippet: Samples were then incubated with rabbit anti-mouse HMGB1 antibody directly conjugated to Alexa 488 (Abcam) for 1 hour at 37°C.

Techniques: Immunofluorescence, Translocation Assay, Staining, Expressing, Derivative Assay, Cell Culture, In Vitro

Journal: Frontiers in Immunology

Article Title: Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation

doi: 10.3389/fimmu.2023.1012799

Figure Lengend Snippet: The combination of histotripsy with checkpoint inhibition results in additive intratumoral infiltration of CD8+ cells and synergistic induction of cancer cell ferroptosis. Mice bearing bilateral B16F10 tumors were treated with no therapy (control), checkpoint inhibition (CI) with anti-CTLA-4 mAb on days 6, 9 and 12 (“CI”), unilateral partial histotripsy ablation (“HT”) on day 7, or both (“HT+CI”). (A) Non-ablated abscopal tumor growth on day 18 was suppressed in mice treated with contralateral HT and CI, but maximal in mice treated with both. (B) Multicolor immunofluorescence of non-ablated abscopal tumors revealed increases in intratumoral CD8+ cell infiltration, loss of nuclear HMGB1, and 4-HNE accumulation after both CI and HT, with maximal effects seen after combinatorial HT+CI. (C) Quantitation of CD8+ cell density demonstrated an additive effect between CI and the abscopal effect of HT. (D) A similar additive effect was observed between CI and the abscopal effect of HT in extranuclear HMGB1 translocation. (E) The combination of HT+CI appeared to be a greater than additive effect on abscopal 4-HNE expression. The additive effects of CI and HT on abscopal CD8+ cell infiltration, HMGB1 release, and 4-HNE expression are shown in line graph form (F) and dot plot form (G) . (n=4-7 mice per group; *=p<0.05 compared with controls; **=p<0.01 compared with controls; ***=p<0.001 compared with controls).

Article Snippet: Samples were then incubated with rabbit anti-mouse HMGB1 antibody directly conjugated to Alexa 488 (Abcam) for 1 hour at 37°C.

Techniques: Inhibition, Immunofluorescence, Quantitation Assay, Translocation Assay, Expressing