mouse ifnγ precoated elispot kit  (Dakewe Biotech Co)

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Rational modulation of tumor microenvironment enhances therapeutic responsiveness to αOX40-based immunotherapy (A–D) OX40-humanized mice bearing subcutaneous MC38 (A), B16 (B), E.G7 (C), or KPC (D) tumors ( n = 5–7 mice per group). Tumor growth curves (numbers indicate complete cures) and Kaplan-Meier survival for each model. Treatments: MPLA+IFN-γ ( i.t. , intratumoral); Combo: MPLA+IFN-γ ( i.t. , intratumoral) + αOX40 ( i.p. , intraperitoneal). (E) Study schema of secondary tumor challenge in MC38 model treated with Combo. (F) Tumor progression and survival outcomes following secondary tumor challenge. Growth kinetics of re-implanted tumors in tumor-cleared mice (previously cured by therapy) versus treatment-naive wild-type controls (left). Kaplan-Meier survival plot (right) ( n = 13 mice per group). (G) Systemic immunity evaluation schema with bilateral MC38 bearing mice were treated with Combo, αOX40, and control. (H) Tumor growth curves and survival plots of (G) ( n = 6–7 mice per group). Data are shown as means ± SD from one of two independent experiments (A–D, F, and H). Statistical significance was determined using log rank test (A–H). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: The antitumor efficacy of the Combo therapy is contingent upon CD8 + T cells and macrophages (A) UMAP of scRNA-seq data from tumor-infiltrating immune cells in OX40-humanized MC38-bearing mice treated with MPLA, IFN-γ, αOX40, or Combo. Cells are color-coded by annotated cell type. (B) Bubble chart showing the top variable marker genes for identified immune cell types. (C) Pie chart shows the relative abundance of 11 immune cell clusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) Macrophage frequency and absolute count in tumors of MC38-bearing mice after two and three treatment cycles with MPLA, IFN-γ, αOX40, or Combo, analyzed by flow cytometry ( n = 5 mice per group). (E) Schematic of CD8 + T cell depletion assay. (F) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study of CD8 + T cell ( n = 6 mice per group). (G) Schematic of macrophage depletion assay in early and late stage. (H and I) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study in (G) ( n = 6–10 mice per group). Data are shown as means ± SD from one of two independent experiments (D, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (D). Log rank test was used (F, H, and I) for statistical comparison. n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Marker, Control, Flow Cytometry, Depletion Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-high macrophages are significantly associated with Combo treatment efficacy (A) UMAP of macrophage subclusters from scRNA-seq data of MC38-bearing mice treated with control, MPLA+IFN-γ, αOX40, or Combo. Cells are color-coded by annotated subtype. (B) Bubble chart showing the top variable marker genes for identified macrophage subclusters. (C) Pie chart shows the relative abundance of four macrophage subclusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) GO pathway analysis identifying significantly enriched signaling pathways in the Mac_S2 subcluster compared to other macrophage subpopulations. (E) Violin plots showing Nos2 expression levels across macrophage subclusters. (F) Violin plots comparing Nos2 and Cd206 expression levels among different treatment groups. (G) Frequency of M1-like, M2-like, or the ratio of M1/M2-like macrophage cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5–10 mice per group). (H) Multiple immunofluorescence signal intensities of NOS2 + F4/80 + and CD206 + F4/80 + cells in the TME of control, αOX40, MPLA+IFN-γ, and Combo groups. Scale bars, 20 μm. Data are shown as means ± SD from one of two independent experiments (G and H). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (G). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Control, Marker, Protein-Protein interactions, Expressing, Flow Cytometry, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-dependent direct tumor cell killing by macrophages in Combo therapy (A) Schematic of co-culture using CFSE-labeled MC38 cells with tumor- or spleen-derived macrophages, with/without NIL treatment. (B) Flow cytometry quantification of 7-AAD + MC38 cells after 48 h co-culture with tumor-(right) or spleen (left)-derived macrophages ( n = 3 biological replicates). (C) Quantification of 7-AAD + MC38 cells after 48 h in vitro co-culture with or without NIL treatment ( n = 3 biological replicates). (D) Treatment schedule for MC38- or B16-tumor-bearing Nos2 KOor WT mice treated with Combo ( n = 6 mice per group). (E and F) Survival curves of MC38-bearing (E) and B16-bearing mice (F) were analyzed using the log rank test. (G) Flow cytometry analysis of 7-AAD + MC38 cells after 48-h co-culture with CFSE-labeled MC38 cells and BMDMs from Nos2 KO mice ( n = 5 biological replicates). (H) Phagocytosis rate of MC38 cells engulfed by BMDMs was assessed by flow cytometry ( n = 3 biological replicates). (I) Surface expression of CRT on MC38 cells was assessed by flow cytometry after co-culture with MPLA- and IFN-γ-polarized BMDMs in vitro ( n = 3 biological replicates). (J) Analysis of CALR + MC38 cells from MC38-tumor-bearing mice following final treatment with control or Combo, assessed by flow cytometry ( n = 5 biological replicates). Data are shown as means ± SD from one of two independent experiments (B, C, E, F, G, H, I, and J). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B, C, and I) or using unpaired Student’s t test (G, H, and J). Log rank tests (E and F) were also used for statistical analysis. n.s., not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (E and F).

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Co-Culture Assay, Labeling, Derivative Assay, Flow Cytometry, In Vitro, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Foxp3 + Treg depletion and macrophage reprogramming are involved in the anti-tumor effect of Combo (A) Flow cytometry analysis of TME. MC38-tumor-bearing mice were treated with control, αOX40, MPLA+IFN-γ, or Combo for two and three doses, and tumors were analyzed by flow cytometry. (B) Frequency and absolute count of CD25 + FOXP3 + cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5 mice per group). (C) Treatment schedule for MC38-tumor-bearing Fcer1g KO or FcγRIIb KO mice. Mice were treated with Control, αOX40, MPLA+IFN-γ, and Combo every 3 days for a total of four doses. (D and E) Survival curves of Fcgr1g KO (D) and FcgrIIb KO (E) mice following treatment ( n = 5–6 mice per group) were monitored. (F) Treatment schedule. MC38-tumor-bearing mice were treated with MPLA and IFN-γ in combination with either OX40-mIgG2a or OX40-hIgG1 agonist antibodies (top), and the corresponding survival curves are shown (bottom) ( n = 5–7 mice per group). (G) Schematic of the co-culture experiment involving BMDMs and Tregs at a ratio of 1:4 (BMDM:Treg); Nos2 expression was measured by RT-qPCR. (H) Relative expression of Nos2 following the co-culture ( n = 4 biological replicates). (I) Multiple immunofluorescence (mIF) staining of MC38 tumors from mice treated with control, αOX40, MPLA+IFN-γ, or Combo, showing FOXP3 and NOS2 expression in border or intra-tumoral. Scale bars, 50 μm. (J) Analysis of cell numbers of FOXP3 and NOS2 expression at the border and intra-tumoral. Representative images from five randomly chosen fields were quantified with ImageJ. Data are shown as means ± SD from one of two independent experiments (B, D, E, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B and H). Log rank test was also used (D–F). n.s., not significant; ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (C–E).

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Flow Cytometry, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF augments cGAS‐STING activation. (a) Quantification of extracellular 2′,3′‐cGAMP in RAW264.7 cell supernatants after 24 h treatment, as measured by enzyme‐linked immunosorbent assay (ELISA) (n = 3). ND, not detected. (b) Immunoblot analysis of the cGAS–STING pathway proteins in RAW264.7 cells treated after 6 h treatment. (c) Quantitative real‐time PCR (qRT–PCR) analysis of IFN‐β mRNA levels in RAW264.7 cells treated with MnSTF for the indicated times (n = 3). (d) Relative expression of IFN‐β mRNA in RAW264.7 cells after 6 h treatment (n = 3). (e–g) ELISA quantification of IFN‐β (e), IL‐6 (f), and TNF‐α (g) in culture supernatants of RAW264.7 cells following 24 h treatment (n = 3). (h) Flow cytometric analysis of CD80 surface expression on DC2.4 dendritic cells after 24 h treatment (n = 3). (i) Representative flow cytometric histograms of MHC class II expression on DC2.4 cells treated for 24 h. Replicates were biological and the data were expressed as mean ± s.d. Statistical analysis was determined using one‐way ANOVA, **** p < 0.0001.

Article Snippet: In the B16F10‐OVA tumor‐bearing mouse model, IFN‐γ ELISpot assays were conducted after treatment using a mouse IFN‐γ precoated ELISpot kit (Dakewe, Cat. 2 210 005).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF regulates the tumor microenvironment by enhancing intratumoral immune responses and reshaping immune cell composition. (a) Representative IVIS fluorescence images of B16F10‐OVA tumor‐bearing mice illustrating intratumoral retention of free ICG or ICG–MnSTF following intratumoral injection. (b) Quantification of intratumoral ICG retention over time (n = 4). (c) Treatment timeline for B16F10‐OVA tumor‐bearing mice administered a single intratumoral injection of PBS or MnSTF (0.69 µmol Mn equivalent), initiated upon tumor palpation (typically 50–100 mm 3 ). (d) Heat map of selected genes in tumors on days 1, 3, and 5 post‐treatment, generated from transcriptome sequencing (n = 3). (e) Tumor IFN‐β levels on day 1 post‐treatment, measured by ELISA (n = 4). (f–i) Representative flow cytometry plots and quantification of F4/80 + iNOS + (f, g) and CD11c + (h, i) cells within tumor tissues at day 1 post‐treatment (n = 4). (j, k) Representative flow cytometry plots (j) and quantification (k) of CD80 + CD86 + expression on CD11c + cells in the draining lymph nodes (LNs) on day 1 post‐treatment (n = 4). (l, m) Representative ELISpot images of IFN‐γ–secreting splenocytes (l) and quantification of spot‐forming units (SFU) per 4 × 10 5 splenocytes (m) on day 7 after three PBS, MnCl 2 , STF, or MnSTF doses (every three days, n = 5). Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using one‐way ANOVA (e, g, i, k, m). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the B16F10‐OVA tumor‐bearing mouse model, IFN‐γ ELISpot assays were conducted after treatment using a mouse IFN‐γ precoated ELISpot kit (Dakewe, Cat. 2 210 005).

Techniques: Fluorescence, Injection, Generated, Sequencing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Enzyme-linked Immunospot

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF synergizes with radiotherapy to enhance immune priming and elicit a robust systemic antigen‐specific T cell response. (a) Schematic illustration of enhanced antitumor immunity induced by RT combined with MnSTF. (b) Experimental timeline for B16F10‐OVA tumor‐bearing mice treated with PBS, RT (6 Gy), MnSTF (0.69 µmol Mn equivalent), or their combination (RT + MnSTF). Treatments were administered every four days for two doses. (c) Tumor growth kinetics following different treatments (n = 9). (d) Tumor IFN‐β levels on day 1 post‐treatments, measured by ELISA (n = 4). (e–h) Representative flow cytometry plots and quantification of CD11c + DCs presenting SIINFEKL–H‐2K b (e, f) and expressing CD80 and CD86 (g, h) in TDLNs on day 1 post‐treatment (n = 4). (i, j) Representative IFN‐γ ELISpot images (i) and quantification of SFU per 5 × 10 5 splenocytes (j) from each group at the end of treatments (n = 5). (k) Representative immunofluorescence images of tumors at treatment end, stained for CD4 + (green) and CD8 + (red) T cells, and DAPI (blue). Scale bar, 120 µm. (n = 3). Replicates were biological and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (c) or one‐way ANOVA (d, f, h, j). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the B16F10‐OVA tumor‐bearing mouse model, IFN‐γ ELISpot assays were conducted after treatment using a mouse IFN‐γ precoated ELISpot kit (Dakewe, Cat. 2 210 005).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Enzyme-linked Immunospot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: mRNA vaccine and MnSTF synergistically potentiate antitumor immune responses. (a) Schematic illustration of enhanced antitumor immunity induced by mRNA vaccine combined with MnSTF. (b) Experimental design schematic in B16F10‐OVA tumor‐bearing mice treated with PBS, MnSTF (0.69 µmol Mn equivalent), LNP‐formulated mRNA OVA (10 µg), or their combination (MnSTF + mRNA OVA ). Treatments were administered every three days for three doses. (c) Tumor growth kinetics after indicated treatments (n = 8). (d, e) Representative flow cytometry plots (d) and quantification (e) of SIINFEKL tetramer + CD8 + T cells in splenocytes collected at the end of treatment (n = 4). (f, h, j, l) Representative IFN‐γ ELISpot images of splenocytes stimulated with SIINFEKL (f), SVYDFFVWL (h), SIYRYYGL (j), or KVPRNQDWL (l) at treatment end. (g,i,k,m) Quantification of IFN‐γ SFU per 2 × 10 5 splenocytes for SIINFEKL (g), or per 10 6 splenocytes for SVYDFFVWL (i), SIYRYYGL (k), and KVPRNQDWL (m), n = 4. Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (c) or one‐way ANOVA (e, g, i, k, m). Significance: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the B16F10‐OVA tumor‐bearing mouse model, IFN‐γ ELISpot assays were conducted after treatment using a mouse IFN‐γ precoated ELISpot kit (Dakewe, Cat. 2 210 005).

Techniques: Flow Cytometry, Enzyme-linked Immunospot

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF acts as a vaccine adjuvant boosting tumor antigen‐specific immunity. (a, b) C57BL/6 mice were subcutaneously immunized with PBS, Alum + OVA (50 µg Alum; 100 µg OVA), or MnSTF + OVA (50 µg Mn; 100 µg OVA) every three days for a total of three doses. Spleens were harvested 7 days after the final immunization to quantify OVA‐specific IFN‐γ–producing splenocytes by ELISpot. Representative IFN‐γ ELISpot images (a) and quantification of SFU per 10 6 splenocytes (b). (c) Schematic of the experimental design in B16F10‐OVA tumor‐bearing mice treated subcutaneously with PBS, MnSTF (0.69 µmol Mn equivalent), OVA (100 µg), or their combination (MnSTF + OVA). Treatments were administered every three days for three doses. (d) Tumor growth kinetics following the indicated treatments (n = 8). (e, f) Representative IFN‐γ ELISpot images (e) and quantification of SFU per 10 6 splenocytes (f), n = 5. (g, h) Representative flow cytometry plots (g) and quantification (h) of effector memory CD44 + CD62L−CD8 + T cells (n = 5). Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (d) or one‐way ANOVA (b, f, h). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the B16F10‐OVA tumor‐bearing mouse model, IFN‐γ ELISpot assays were conducted after treatment using a mouse IFN‐γ precoated ELISpot kit (Dakewe, Cat. 2 210 005).

Techniques: Adjuvant, Enzyme-linked Immunospot, Flow Cytometry