invivoplus rat igg2a isotype control clone: 2a3 catalog #: bp0089 lot #627416n1  (Bio X Cell)

Journal: Cell Reports Medicine

Article Title: Fc-optimized anti-CTLA-4 antibodies increase tumor-associated high endothelial venules and sensitize refractory tumors to PD-1 blockade

doi: 10.1016/j.xcrm.2025.102141

Figure Lengend Snippet: Fc-enhanced mouse anti-CTLA-4 antibodies prune TA-HEVs in the absence of concurrent PD-1 blockade (A) Treatment schedule. i.p., intraperitoneal; control, isotype control antibodies. (B and C) Mean and individual tumor growth. Data were obtained from two independent experiments ( n = 10 mice per group). (D–G) Frequency of FOXP3 + Tregs in tumor, numbers of tumor-infiltrating FOXP3 + Tregs and FOXP3 − Tconv, and ratio of Tconv to Treg cell numbers in tumors following indicated treatments. Each symbol represents an individual mouse ( n = 10, two independent experiments). (H–M) Frequencies of CD45 − CD31 high endothelial cells, TA-HECs, MECA-79 + CD62P + TA-HECs, numbers of TA-ECs and TA-HECs, and mean fluorescence intensity (MFI) of MECA-79 and CD62P in TA-HECs, following indicated treatments, quantified by flow cytometry. Each symbol represents an individual mouse ( n = 10, two independent experiments). (N) Histograms showing expression of PD-L1 in CD45 − CD31 high tumor endothelial cells following indicated treatments, quantified by flow cytometry. MFI of PD-L1 is quantified. Each symbol represents an individual mouse ( n = 4–5). (O) Treatment schedule. i.p., intraperitoneal; control, isotype control antibodies. (P and Q) Mean and individual tumor growth. Data were obtained from two independent experiments (control, n = 8 mice; 9D9-IgG2a + anti-PD-1, n = 10 mice). (R–T) Numbers of tumor-infiltrating FOXP3 + Tregs and FOXP3 − Tconv, and ratio of Tconv to Tregs cell numbers in tumors following indicated treatments. Each symbol represents an individual mouse ( n = 8–10, two independent experiments). (U) Frequency of TA-HECs. Representative dot plots of TA-HECs are shown. Each symbol represents an individual mouse ( n = 8–10, two independent experiments). Data are shown as mean ± SEM. All p values were determined by unpaired two-tailed Student’s t test except for graph in (N) for which p values were determined by one-way ANOVA with Tukey’s multiple comparison test, and for (B) and (P) for which p values were determined by two-way ANOVA. See also .

Article Snippet: InVivo MAb Rat IgG2a isotype control (clone 2A3) , Bio X Cell , Cat# BE0089; RRID: AB_1107769.

Techniques: Control, Fluorescence, Flow Cytometry, Expressing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Mass spectrometry analysis of CD11b immunoprecipitates from BMDMs. Left: experimental workflow for isolating the CD11b-interacting proteins. Right: relative abundance of CD11b-interacting proteins, with a focus on cell surface receptors with inhibitory potential, indicated by a color gradient. ITIM, immunoreceptor tyrosine-based inhibitory motif; ITSM, immunoreceptor tyrosine-based switch motif; NPXY, asparagine, proline, any residue, tyrosine. IP, immunoprecipitation. b Immunoblot analysis of CD11b and CD200R1 interaction in immunoprecipitates from WT, Itgb2 −/− and Cd200r1 −/− BMDMs. Relative abundance is shown at the bottom of each panel. c Flow cytometry analysis of CD200 expression on parental (top) and Tac expression on Tac + (bottom) mouse tumor cell lines. Red curves represent staining with CD200 or Tac mAbs. Filled curves, control (Ctrl) mAbs. d Microscopy-based phagocytosis assay of non-opsonized (−) or IgG-opsonized (+) Tac + WEHI-231, Tac + A20, Tac + J558 and Tac + TUBO cells by WT BMDMs, in presence of CD200 mAb OX-90 (rat IgG2a) or Ctrl mAb 2A3 (rat IgG2a). Tumor cells were opsonized with Tac mAb 7G7 (mouse IgG2a), ( n = 3). e Time-course pHrodo-based phagocytosis assay using IgG-opsonized Tac + WEHI-231 cells and WT BMDMs. WEHI-231 cells were labeled with pHrodo red dye, and BMDMs were labeled with CSFE. Phagocytosis over time (0-4 h) was analyzed using an IncuCyte Live Cell Analyzer. Left: representative images at 2 h (scale bar, 100 μm; arrows, BMDMs with engulfed tumor cells). Right: quantification of cumulative phagocytosis (top) and time-specific increase in phagocytosis (Δ phagocytosis; bottom) over 0-4 h, ( n = 3). f Confocal microscope-based conjugate formation and actin polarization assay of IgG-opsonized WEHI-231 cells labeled with CSFE (green) and co-incubated with WT BMDMs labeled with Cell Trace Violet (CTV; blue), in presence of CD200 mAb or Ctrl mAb. Actin (red) was detected by β-actin mAb. Left: representative images (scale bar, 10 μm; arrows, BMDMs with fully polarized actin). Scale, 10 μm. Right: quantification of conjugate formation (top) and of conjugates with fully polarized actin (bottom), ( n = 3). g As per Fig. 1d, except that phagocytosis of complement (C3bi)-opsonized WEHI-231 cells by WT BMDMs in the presence of blocking CD200 mAb OX-90, blocking CD11b mAb 5C6 (rat IgG2b), Ctrl mAb 2A3 or Ctrl mAb LTF-2 (rat IgG2b), ( n = 3). h As per Fig. 1d, expect that phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs in the presence of CD200R1 mAb OX-131 (mouse IgG1, Fc-silent) or Ctrl mAb (mouse IgG1, Fc-silent), ( n = 3). Data are from three ( a – h ) independent experiments. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t -test ( d – f , h ) with multiple comparisons ( e ); One-way ANOVA test with multiple comparisons ( g ); ns, not significant. See also Supplementary Fig. and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Mass Spectrometry, Residue, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing, Staining, Control, Microscopy, Phagocytosis Assay, Labeling, Incubation, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200R1 expression (left) on WT (top) or Cd200r1 −/− (bottom) BMDMs, and CD200 expression (right) on Cd200 +/+ (top) or Cd200 −/− (bottom) WEHI-231 cells, as measured by flow cytometry. Red curves, CD200R1 or CD200 mAbs. Filled curves, Ctrl mAb. b As per Fig. , except that phagocytosis of IgG-opsonized WEHI-231 cells in the presence of WT or Cd200r1 −/− BMDMs was studied, ( n = 3). c As per Fig. 2b, except that phagocytosis of IgG-opsonized Cd200 +/+ and Cd200 −/− WEHI-231 cells, in the presence of WT or Cd200r1 −/− BMDMs, ( n = 3). Data are from three independent experiments ( a – c ). Each symbol represents one mouse. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , c ), ns not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (top) or tumor antigens (CD20, CD38, CD123, DLL3 and SLAMF7; bottom) on various human tumor cell lines. Red curves, CD200 mAb or tumor antigen-specific mAbs. Filled curves, Ctrl mAbs. b As per Fig. , except using IL-4-primed human blood monocyte-derived macrophages and human tumor cells in the presence of samalizumab (human IgG1, Fc-silent) or Ctrl mAb MOPC21 (human IgG1, Fc-silent). IgG opsonization was performed using CD20 mAb rituximab (SLVL, 721.221), CD38 mAb daratumumab (NCI-H929), CD123 mAb talacotuzumab (KG-1a), DLL3 mAb rovalpituzumab (NCI-H209), or SLAMF7 mAb elotuzumab (SK-MEL-28) ( n = 3 or 4). c As per Fig. 5b, except using the indicated cell lines and samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent), or Ctrl mAb MOPC21 (human IgG1, Fc-silent). Tumor cells were opsonized as detailed for Fig. 5b, ( n = 3). d –f Subcutaneous injection of SLVL cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAb every 2 days, ( n = 8). d Schematic representation of the experimental workflow. e Tumor volume over time. f Tumor weight. g –i Subcutaneous injection of 721.221 cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAbs every 2 days, ( n = 10). g Schematic representation of the experimental workflow. h Tumor volume over time. i Tumor weight. Data are from three to four ( a, b ), three ( c ), or two ( d-i ) independent experiments. Each symbol represents one human sample or one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t-test ( f , i ), with multiple comparisons ( b, c , e , h ). ns, not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Derivative Assay, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Partial sequences of the cytoplasmic domain of CD200R1 from different species. The conserved tyrosines (Y 286 , Y 289 , and Y 297 ; based on mouse amino acid numbering) are in red, whereas the conserved NPxY motif is boxed. Identical residues are depicted by asterisks (*), while conserved and semi-conserved amino acids are highlighted by colons (:) and periods (.), respectively. b Phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs expressing GFP alone, or Cd200r1 −/− BMDMs expressing GFP alone or CD200R1 variants, in the presence of CD200 mAb or Ctrl mAb, ( n = 3). c Mass spectrometry analyzes of cytoplasmic proteins with inhibitory potential interacting with synthetic biotinylated CD200R1 peptides, with or without phosphorylation at Y 286 or Y 297 in pull-down assays. Peptides are depicted at the top. Interactors identified by phosphorylated peptides are shown below. Negative regulators of immune cell activation, either adaptors, kinases, phosphatases or Ras-GAP, are indicated. d Immunoblot analysis of Dok-1 and Dok-2 expression (left) and phagocytosis of IgG-opsonized WEHI-231 cells (right) by BMDMs from WT or Dok1 −/− Dok2 −/− mice. β-actin as loading Ctrl (left). Normalized protein abundance (in %) relative to actin is shown below the top panel, ( n = 4). e Same as Fig. 6d except that WT BMDMs transduced with Ctrl or Csk-specific siRNAs [ Csk knockdown ( Csk KD )] were used. Two different Csk-specific siRNAs were studied. Csk expression (left) and phagocytosis (right) were studied, ( n = 4). f Summary of fold-changes in phagocytosis for the various genetically deficient BMDMs in response to CD200 mAb, compared to WT BMDMs, ( n = 3 or 4). g WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 1 min with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were immunoprecipitated with α-Dok-1, α-Dok-2, α-Csk, or normal rabbit serum (NRS), and probed by immunoblotting with antibodies targeting phosphotyrosine (pTyr), Dok-1, Dok-2 or Csk. h WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 30 s with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were probed with α-pLyn (Tyr 507) or α-Lyn Abs (top). A quantification of multiple independent experiments is shown at the bottom, ( n = 3). Data are from three ( b , e , g , h ) or four d independent experiments, two (pY 297 peptide) and three (pY 286 peptide) ( c ) independent experiments. Each symbol represents one mouse ( b , d , e ). Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , d , e , h ); One-way ANOVA test, with multiple comparisons ( f ). ns not significant. See also Supplementary Figs. , and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Expressing, Mass Spectrometry, Phospho-proteomics, Activation Assay, Western Blot, Quantitative Proteomics, Transduction, Knockdown, Immunoprecipitation

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (red curves; top) and CD47 (lavender curves; bottom) expression on J558, A20 and WEHI-231 cells (left). Filled curves, Ctrl mAbs. The right panel shows relative expression levels of CD200 and CD47. b As per Fig. , except that mAbs were used in combination: CD200 mAb OX-90 (rat IgG2a), SIRPα mAb 27 (mouse IgG2a, Fc-silent), Ctrl mAb 2A3 (rat IgG2a), and Ctrl mAb MOPC21 (mouse IgG2a, Fc-silent), ( n = 3). c – f Luciferase + Tac + GFP + WEHI-231 cells were injected intravenously into Rag1 −/− mice, followed by intraperitoneal injection of Tac mAb combined with the indicated mAbs every 2 days starting from day 4, ( n = 5). c Schematic representation of the experimental workflow. Tumor progression was measured over time using luminescence. Representative photographs of mice ( d ) and quantification ( e ). f Kaplan – Meier curves of survival. g Phagocytosis of normal activated human T cells or B cells by autologous human macrophages, in the presence of samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent) or Ctrl mAb (human IgG1, Fc-silent). T cells were not opsonized (implying phagocytosis was mediated by SLAMF7), whereas B cells were opsonized with rituximab (human IgG1, Fc-active) ( n = 3). Data are from three ( a , b , g ) or two ( c – f ) independent experiments. Each symbol represents one mouse or donor. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , e ); log-rank (Mantel-Cox) test ( f ); one-way ANOVA test, with multiple comparisons ( g ). ns, not significant.

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing, Luciferase, Injection

Journal: iScience

Article Title: Trajectories of macrophage ontogeny and reprogramming in cancer

doi: 10.1016/j.isci.2025.112498

Figure Lengend Snippet: D KO enhances tumor response to antiangiogenic immunotherapy (A) Schematic of the experimental setup. (B) Volume of individual lung tumors (mean ± SEM) in mice treated as indicated and imaged by micro-CT at day 28 post-tumor injection. n=6 D WT mice treated with IgG1+IgG2a (WT IgGs); n=7 D WT mice treated with A2V+α-PD1 (WT A2V+α-PD1); n=5 D KO mice (KO IgGs and KO A2V+α-PD1). Note that each mouse carries several independent lung tumors. Statistical analysis by unpaired Student’s t test performed between control and interventional treatment for each independent genotype. ∗ p ≤ 0.05.

Article Snippet: Rat IgG2a Isotype Control; clone 2A3 , BioXCell , Cat#BE0089 RRID: AB_1107769.

Techniques: Micro-CT, Injection, Control

Journal: iScience

Article Title: Trajectories of macrophage ontogeny and reprogramming in cancer

doi: 10.1016/j.isci.2025.112498

Figure Lengend Snippet: D KO enhances tumor response to antiangiogenic immunotherapy (A) Schematic of the experimental setup. (B) Volume of individual lung tumors (mean ± SEM) in mice treated as indicated and imaged by micro-CT at day 28 post-tumor injection. n=6 D WT mice treated with IgG1+IgG2a (WT IgGs); n=7 D WT mice treated with A2V+α-PD1 (WT A2V+α-PD1); n=5 D KO mice (KO IgGs and KO A2V+α-PD1). Note that each mouse carries several independent lung tumors. Statistical analysis by unpaired Student’s t test performed between control and interventional treatment for each independent genotype. ∗ p ≤ 0.05.

Article Snippet: For the latter study, irrelevant control IgGs were a combination of IgG1 (20 mg/kg; clone MOPC-21, Roche) and rat IgG2a (10 mg/kg; clone 2A3, BE0089, Bio X Cell).

Techniques: Micro-CT, Injection, Control