elisa kits for dock8  (MyBiosource Biotechnology)

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release HMGB1 as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Injection, Activation Assay, Expressing, Inhibition

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 1. Celastrol (CEL) induces ICD and down-regulates PD-L1 expression in melanoma in vitro and in vivo. (A) Chemical structure of CEL. (B) CEL triggered autophagy in melanoma cells. Mouse B16F10 or BPD6 melanoma cells were incubated with CEL (0.1–8 μM) for 12 h and then lysed for Western blot analysis of LC3B, whose subunit transition from LC3B I to LC3B II is a marker for activated autophagy. (C) Immunofluorescent imaging of CRT, HMGB1, and PD-L1 in mouse (B16F10 and BPD6) and human (M10 and A375) melanoma cells after treated with CEL at 1 μM; The treatment time was 4 h for CRT and 24 h for HMGB1 and PD-L1 detection. Cell nuclei were stained with DAPI. Scale bar indicates 20 μm. Each value was quantified in 5 randomly selected fields. Each sample was repeated 3 times. (D) Flow cytometry analysis of CRT+ melanoma cells after treated with CEL or positive control, doxorubicin (DOX) and mitoxantrone (MIT), for 4 h at their IC50. (E) The released HMGB1 in the cell culture medium 24 h after incubation with CEL, DOX or MIT at their IC50 doses (n = 4). (F) RT-PCR analysis of the PD-L1 mRNA levels in B16F10 and BPD6 cells after incubation with CEL, DOX or MIT at IC50 for 24 h (n = 6). (G) Western blot analysis of PD-L1 expression in tumors at 48 h after i.t. injection with CEL (0.15 mg/kg), DOX (0.01 mg/kg) or MIT (0.5 mg/kg) or i.p. injection with αPD-L1 (5 mg/kg). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Expressing, In Vitro, In Vivo, Incubation, Western Blot, Marker, Imaging, Staining, Flow Cytometry, Positive Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 2. Characterization of celastrol nanoemulsion (CEL NE) and its ICD-induction and PD-L1 downregulation on melanoma. (A) The size distribution of CEL NE characterized by dynamic laser light scattering and transmission electron microscopy (insets). (B) The dose-dependent curve of CRT+-population of B16F10 cells incubated with free CEL or CEL NE for 4 h. The percentage of CRT+ cells was quantified as an average of 5 randomly selected fields. Each sample was repeated 3 times; see Supplementary Fig. S4 for the curves of other cells. (C–F) Analysis of in vivo ICD-induction and PD-L1 downregulation. The mice bearing B16F10 tumors received a single i.t. injection of CEL NE or free CEL (0.15 mg/kg) (n = 4) and were sacrificed after 48 h for analysis: (C) Representative immunofluorescent images of CRT, HMGB1 and activated DC markers (CD86, CD11c, and MHC II) in B16F10 tumors after i.t. injection of CEL NE or free CEL. Each value was quantified as an average of 5 randomly selected fields. Scale bar indicates 300 μm. (D) Statistic analysis of co-stimulatory markers, CD80 and CD86, in the draining lymph nodes by flow cytometry (n = 4). (E) Flow cytometry quantitation of PD-L1+ melanoma cell percentages (PD-L1+MART+) in the tumors. (F) RT-PCR analysis of PD-L1 mRNA levels in the tumors. (G) Western blot analysis of dose-dependent downregulation of NF-κB and PD-L1 in the tumors. The mice with tumors as above were i.t. injected with the indicated doses of CEL NE and analyzed after 48 h (n = 4). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Transmission Assay, Electron Microscopy, Incubation, In Vivo, Injection, Flow Cytometry, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot