invivomab anti-mouse cd16/cd32 clone 2.4g2  (Bio X Cell)

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Induction of anti-tumor immune responses by various adjuvanted OVA (A–E) Schematic of representation of the experimental design. Naive female C57BL/six mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached to approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 4, and 7 (A). Average growth curves shown are mean ± SEM from two independent experiments (PBS, n = 8; OVA, n = 8; Alum + OVA, n = 8; MF59 + OVA, n = 8; CR108 + OVA, n = 9) (B). Statistical significances: # (CR108 + OVA vs. Alum + OVA, p < 0.05), ##(CR108 + OVA vs. Alum + OVA, p < 0.01) ∗(CR108 + OVA vs. MF59 + OVA, p < 0.05), and ns (CR108 + OVA vs. MF59 + OVA, not significant) by Student’s t test (C). The tumor weights were determined for each group. Quantification of tumor-infiltrating CD45 + CD3 + CD8 + T cells (D), and CD45 + CD3 + CD4 + T cells were determined by FACS at the end of the experiment (E) ( A). Data shown are representative of two independent experiments with four mice in each experiment (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, D, E). (F) In vivo CTL response measurement: C57BL/six mice were subcutaneously immunized with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS on days 0 and 14. On day 21 post-first immunization. Cells pulsed with OVA 257–264 peptides were stained with CFSE high , while unpulsed cells were stained with CFSE low . CFSE high , and CFSE low cells were mixed in 1:1 and then intravenously transferred into the immunized mice. Twenty hours later, splenocytes were analyzed by flow cytometry to assess OVA-specific lysis. Data shown are representative of two independent experiments with 5–6 mice in each experiment (PBS, n = 6; OVA, n = 6; Alum + OVA, n = 6; MF59 + OVA, n = 6; CR108 + OVA, n = 5) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by One-way ANOVA. (G–I) Schematic representation of the treatment experimental design. Naive female C57BL/6 mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 7, and 14 (G). Average tumor growth curves (H) and the survival rates were measured until day 17 (I). Data shown are mean ± SEM from two independent experiments (untreated, n = 9; OVA, n = 10; Alum + OVA, n = 9; MF59 + OVA, n = 10; CR108 + OVA, n = 9). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (H). (J–L)Experimental design for α-CD4 or α-CD8 blocking. naive female C57BL/six mice were inoculated s.c. with 5 × 10 5 E.G7-OVA cells in the right flank. Once tumors reached 20 mm 3 , mice were intraperitoneally (i.p.) treated with 100 μg α-CD4 (GK1.5, BioXcell), 100 μg α-CD8 (2.43, BioXcell), or 100 μg rat IgG2b isotype control (LTF-2, BioXcell) on days 0, 5, 10, and 15. CR108 + OVA or PBS (untreated control) was administered near the tumor site on days 1, 7, and 13 (J). Average tumor growth curves (K) and survival rates (L) were determined. Data shown are mean ± SEM from two independent experiments (α-CD8 CR108 + OVA, n = 7; α-CD4 CR108 + OVA, n = 8; isotype CR108 + OVA, n = 7). Statistical significance: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (K).

Article Snippet: InVivoMAb Rat anti-mouse CD4 IgG2b; Clone GK1.5 , BioXcell, West Lafayette, IN, USA , Cat# BE0003-1, RRID: AB_1107636.

Techniques: Control, In Vivo, Staining, Flow Cytometry, Lysis, Blocking Assay

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Loss of CD8 + T cell activation due to CCR7 blockage affecting Mig DCs recruitment (A–C) Experimental procedure: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, 10 μg of α-CCR7 (4B12, R&D) blocking antibody per mouse was injected subcutaneously at the photoconverted skin site, while 10 μg of rat IgG2a antibody (2A3, BioXcell) per mouse was used as an isotype control. Six hours later, CR108 or MF59 plus OVA were administered subcutaneously at the α-CCR7 injection site. Forty-eight hours after administration, the numbers of (A) MHCII hi CD11c med Mig DCs, (B) KikGR-red cells, and (C) MHCII med CD11c hi Res DCs in the dLNs were measured by FACS. Data shown are representative of two independent experiments with three mice in each experiment (mean ± SEM). ∗ p < 0.05, ns (not significant) by Student’s t test (A, B, C). (D–H) Schematic of Experimental Design: CFSE-labeled OTI CD8 + T cells (1 × 10 6 ) were transferred into naive C57BL/six mice. One day later, the mice were subcutaneously injected with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control. Subsequently, the mice were immunized at the antibody-injected site with CR108 or MF59 plus OVA, and PBS as a vehicle control. Three days post-immunization, CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells in dLNs, non-dLNs, and spleens were analyzed by FACS (D). Histogram analysis: histogram overlays of CFSE-dilution in CD45.1 + TCRVα2 + CD8 + T cells from dLNs, non-dLNs, and spleens were presented. (E) Quantitative Analysis: The number of CFSE lo CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) was quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (mean ± SEM) (isotype CR108 + OVA, n = 4; α-CCR7 CR108 + OVA, n = 4; isotype MF59 + OVA, n = 3; α-CCR7 MF59 + OVA, n = 4; PBS, n = 4). ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, not significant by Student’s t test (F, G, H). (I) Experimental design for α-CCR7 blocking: naive female C57BL/six mice were inoculated subcutaneously with E.G7-OVA cells (1 × 10 6 ) in the right flank, and tumor growth was monitored until the volume reached 20 mm 3 . To block Mig DCs migration, mice bearing E.G7-OVA tumors were treated subcutaneously with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control on days 1, 7, and 13. CR108 plus OVA were administered near the tumor sites, and PBS as a vehicle control (untreated), on days 1, 7, and 13. Tumor Growth Curves: Average tumor growth curves (α-CCR7 CR108 + OVA, n = 5; isotype CR108 + OVA, n = 4) were determined. Data shown are mean ± SEM from two independent experiments. ∗ p < 0.05 by two-way ANOVA.

Article Snippet: InVivoMAb Rat anti-mouse CD4 IgG2b; Clone GK1.5 , BioXcell, West Lafayette, IN, USA , Cat# BE0003-1, RRID: AB_1107636.

Techniques: Activation Assay, Blocking Assay, Injection, Control, Labeling, Migration

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Activation of CD8 + T cells primed by adjuvanted OVA via activated KikGR-red cells (A) Experimental design: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, the photoconverted skin site was treated with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Forty-eight hours post-administration, KikGR-red cells, MHCII hi CD11c med Mig DCs, and MHCII med CD11c hi Res DCs were isolated from the dLNs using FACS sorting. Naive OT-I CD8 + T cells at 5 × 10 4 per well isolated from OT-I mice were used to co-culture with either 5 × 10 3 of KikGR-red, MHC-II hi CD11c med Mig DCs, or MHC-II med CD11c hi Res DCs per well, respectively. (B–D) CD8 + T cell activation: three days post-co-culture, the number of CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells co-cultured with KikGR-red cells (B), MHC-II hi CD11c med Mig DCs (C), or MHC-II med CD11c hi Res DCs (D) was measured by FACS. Data shown in (B) and (D) are representative of two independent experiments with 3–4 mice in each experiment (Alum + OVA, n = 4; MF59 + OVA, n = 3; CR108 + OVA, n = 4) (mean ± SEM). Data shown in (C) are representative of two independent experiments with 6–8 mice in each experiment (Alum + OVA, n = 7; MF59 + OVA, n = 8; CR108 + OVA, n = 6) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (B, C, D).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Control, Isolation, Co-Culture Assay, Cell Culture

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Activation of CD8 + T Cells following immunization with various adjuvanted OVA (A) Experimental design: CFSE-labeled OT-I CD3 + CD8 + T cells (1 × 10 6 ) were isolated from donor animals and adoptively transferred intravenously into naive CD45.2 C57BL/six mice on day 0. The recipient mice were then immunized subcutaneously (s.c.) on day 1 with one of the following: CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Three days post-immunization, CD45.1 + TCRVα2 + CD8 + T cell proliferation was assessed within draining lymph nodes (dLNs), non-draining lymph nodes (non-dLNs), and spleen via CFSE-dilution using flow cytometry. (B, C, and D) CFSE-dilution analysis in dLNs: histogram overlays depict CFSE-dilution of CD45.1 + TCRVα2 + CD8 + T cells in dLNs for each immunization group, illustrating the extent of T cell proliferation (B). (C) Proliferation index calculation: The T cell proliferation index was determined to quantify the overall proliferation response across different immunizations. (D) Proliferation in CFSE-division peaks: the percentage of CD45.1 + TCRVα2 + CD8 + T cells in each CFSE-division peak within dLNs was analyzed. Statistical significances: # p < 0.05, and #### p < 0.0001 (CR108 + OVA versus Alum + OVA), ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (CR108 + OVA versus MF59 + OVA) by Student’s t test, respectively. (E) Proliferative profiles in lymphoid organs: histogram overlays showing CFSE-dilution profiles of CD45.1 + TCRVα2 + CD8 + T cells in dLNs, non-dLNs, and spleens for all experimental groups, highlighting the differential T cell proliferation across these tissues. (F, G, and H) CD8 T cell proliferation in lymphoid tissues: the percentages of CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) were quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (PBS, n = 3; OVA, n = 4; Alum + OVA, n = 4; MF59 + OVA, n = 4; CR108 + OVA, n = 4) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, F, G, H).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Labeling, Isolation, Control, Flow Cytometry

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Induction of anti-tumor immune responses by various adjuvanted OVA (A–E) Schematic of representation of the experimental design. Naive female C57BL/six mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached to approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 4, and 7 (A). Average growth curves shown are mean ± SEM from two independent experiments (PBS, n = 8; OVA, n = 8; Alum + OVA, n = 8; MF59 + OVA, n = 8; CR108 + OVA, n = 9) (B). Statistical significances: # (CR108 + OVA vs. Alum + OVA, p < 0.05), ##(CR108 + OVA vs. Alum + OVA, p < 0.01) ∗(CR108 + OVA vs. MF59 + OVA, p < 0.05), and ns (CR108 + OVA vs. MF59 + OVA, not significant) by Student’s t test (C). The tumor weights were determined for each group. Quantification of tumor-infiltrating CD45 + CD3 + CD8 + T cells (D), and CD45 + CD3 + CD4 + T cells were determined by FACS at the end of the experiment (E) ( A). Data shown are representative of two independent experiments with four mice in each experiment (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, D, E). (F) In vivo CTL response measurement: C57BL/six mice were subcutaneously immunized with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS on days 0 and 14. On day 21 post-first immunization. Cells pulsed with OVA 257–264 peptides were stained with CFSE high , while unpulsed cells were stained with CFSE low . CFSE high , and CFSE low cells were mixed in 1:1 and then intravenously transferred into the immunized mice. Twenty hours later, splenocytes were analyzed by flow cytometry to assess OVA-specific lysis. Data shown are representative of two independent experiments with 5–6 mice in each experiment (PBS, n = 6; OVA, n = 6; Alum + OVA, n = 6; MF59 + OVA, n = 6; CR108 + OVA, n = 5) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by One-way ANOVA. (G–I) Schematic representation of the treatment experimental design. Naive female C57BL/6 mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 7, and 14 (G). Average tumor growth curves (H) and the survival rates were measured until day 17 (I). Data shown are mean ± SEM from two independent experiments (untreated, n = 9; OVA, n = 10; Alum + OVA, n = 9; MF59 + OVA, n = 10; CR108 + OVA, n = 9). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (H). (J–L)Experimental design for α-CD4 or α-CD8 blocking. naive female C57BL/six mice were inoculated s.c. with 5 × 10 5 E.G7-OVA cells in the right flank. Once tumors reached 20 mm 3 , mice were intraperitoneally (i.p.) treated with 100 μg α-CD4 (GK1.5, BioXcell), 100 μg α-CD8 (2.43, BioXcell), or 100 μg rat IgG2b isotype control (LTF-2, BioXcell) on days 0, 5, 10, and 15. CR108 + OVA or PBS (untreated control) was administered near the tumor site on days 1, 7, and 13 (J). Average tumor growth curves (K) and survival rates (L) were determined. Data shown are mean ± SEM from two independent experiments (α-CD8 CR108 + OVA, n = 7; α-CD4 CR108 + OVA, n = 8; isotype CR108 + OVA, n = 7). Statistical significance: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (K).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Control, In Vivo, Staining, Flow Cytometry, Lysis, Blocking Assay

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Loss of CD8 + T cell activation due to CCR7 blockage affecting Mig DCs recruitment (A–C) Experimental procedure: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, 10 μg of α-CCR7 (4B12, R&D) blocking antibody per mouse was injected subcutaneously at the photoconverted skin site, while 10 μg of rat IgG2a antibody (2A3, BioXcell) per mouse was used as an isotype control. Six hours later, CR108 or MF59 plus OVA were administered subcutaneously at the α-CCR7 injection site. Forty-eight hours after administration, the numbers of (A) MHCII hi CD11c med Mig DCs, (B) KikGR-red cells, and (C) MHCII med CD11c hi Res DCs in the dLNs were measured by FACS. Data shown are representative of two independent experiments with three mice in each experiment (mean ± SEM). ∗ p < 0.05, ns (not significant) by Student’s t test (A, B, C). (D–H) Schematic of Experimental Design: CFSE-labeled OTI CD8 + T cells (1 × 10 6 ) were transferred into naive C57BL/six mice. One day later, the mice were subcutaneously injected with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control. Subsequently, the mice were immunized at the antibody-injected site with CR108 or MF59 plus OVA, and PBS as a vehicle control. Three days post-immunization, CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells in dLNs, non-dLNs, and spleens were analyzed by FACS (D). Histogram analysis: histogram overlays of CFSE-dilution in CD45.1 + TCRVα2 + CD8 + T cells from dLNs, non-dLNs, and spleens were presented. (E) Quantitative Analysis: The number of CFSE lo CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) was quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (mean ± SEM) (isotype CR108 + OVA, n = 4; α-CCR7 CR108 + OVA, n = 4; isotype MF59 + OVA, n = 3; α-CCR7 MF59 + OVA, n = 4; PBS, n = 4). ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, not significant by Student’s t test (F, G, H). (I) Experimental design for α-CCR7 blocking: naive female C57BL/six mice were inoculated subcutaneously with E.G7-OVA cells (1 × 10 6 ) in the right flank, and tumor growth was monitored until the volume reached 20 mm 3 . To block Mig DCs migration, mice bearing E.G7-OVA tumors were treated subcutaneously with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control on days 1, 7, and 13. CR108 plus OVA were administered near the tumor sites, and PBS as a vehicle control (untreated), on days 1, 7, and 13. Tumor Growth Curves: Average tumor growth curves (α-CCR7 CR108 + OVA, n = 5; isotype CR108 + OVA, n = 4) were determined. Data shown are mean ± SEM from two independent experiments. ∗ p < 0.05 by two-way ANOVA.

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Blocking Assay, Injection, Control, Labeling, Migration