Journal: Frontiers in Immunology

Article Title: Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice

doi: 10.3389/fimmu.2024.1432816

Figure Lengend Snippet: Antibodies used in the myeloid and lymphoid panels.

Article Snippet: CD172a , VioBlue , Miltenyi , 0.5μL , 130-123-151 , REA1201.

Techniques:

Journal: Frontiers in Immunology

Article Title: Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice

doi: 10.3389/fimmu.2024.1432816

Figure Lengend Snippet: Intracellular panel at 72 hours post-injection (p.i.). This figure shows relative counts from the Intracellular panel in animals treated with LPS and in control animals at 72 hours post-injection. Only populations with significant differences between groups are presented. Significance levels are indicated as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (A) CD45 Leukocytes; (B) Monocyte type myeloid derived suppressor cell; (C) Lymphocytes; (D) Tcells; (E) CD4 Tcells; (F) CD4 Effector memory; (G) CD4 CD44 positive; (H) T Helper 9 Cells; (I) CD8 Tcells; (J) CD8 Central memory; (K) CD8 Effector activated; (L) lymphocytes B; (M) Natural Killers; (N) CD8 Effector memory; (O) plasma cells; (P) Natural killer T cell; (Q) CD172a myeloids cells.

Article Snippet: CD172a , VioBlue , Miltenyi , 0.5μL , 130-123-151 , REA1201.

Techniques: Injection, Control, Derivative Assay, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice

doi: 10.3389/fimmu.2024.1432816

Figure Lengend Snippet: Proportions of variance (ƞ2) associated with intra- and inter-individual factors.

Article Snippet: CD172a , VioBlue , Miltenyi , 0.5μL , 130-123-151 , REA1201.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice

doi: 10.3389/fimmu.2024.1432816

Figure Lengend Snippet: Effect size corresponding to a Cohen’s d of 0.5 for the overall variables studied in the myeloid and lymphoid panels.

Article Snippet: CD172a , VioBlue , Miltenyi , 0.5μL , 130-123-151 , REA1201.

Techniques: Clinical Proteomics

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) expression is downregulated in dilated cardiomyopathy (DCM) human hearts and aortic banding (AB)-operated mouse hearts. A and B, Protein levels of β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and SIRPA in samples from donor hearts and DCM hearts (n=3; *P<0.05 vs donor hearts). C and D, Protein levels of β-MHC, ANP, and SIRPA in samples from wild-type mice at the indicated times after sham or AB surgery (n=3; *P<0.05 vs sham). E and F, Protein levels of β-MHC, ANP, and SIRPA in samples from neonatal rat cardiomyocytes treated with angiotensin II (Ang II; 1 μmol/L) or phenylephrine (PE; 100 μmol/L) for 48 h (n=3; *P<0.05 vs PBS). Representative blots (A, C, and E) and quantitative results (B, D, and F). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) deficiency augments pressure overload–induced cardiac hypertrophy and fibrosis. A, Images of heart sections stained with H&E and WGA from wild-type (WT) and knockout mice 4 weeks after sham or aortic banding (AB) surgery (n=6–10 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=13–15 mice per experimental group). F, Images of heart sections stained with picrosirius red from WT and knockout mice 4 weeks after sham or AB surgery (n=6–10 mice per experimental group). G, Statistical results for the left ventricular (LV) collagen volume (%; n=>40 fields). *P<0.05 vs WT/sham; #P<0.05 vs WT/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Staining, Knock-Out

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α overexpression mitigates aortic banding (AB)-induced cardiac remodeling. A, Images of heart sections stained with H&E and WGA from nontransgenic (NTG) and transgenic (TG) mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=14–15 mice per experimental group). F, Images of heart sections stained with PSR from NTG and TG mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). G, Statistical results for the left ventricular collagen volume (%; n=>40 fields). *P<0.05 vs NTG/sham; #P<0.05 vs NTG/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Over Expression, Staining, Transgenic Assay

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) inhibits the Toll-like receptor 4 (TLR4) signaling pathway. A and B, Protein expression levels of TLR4 are upregulated in the SIRPA knockout mice but downregulated in the SIRPA transgenic mice 2 weeks after aortic banding (AB) surgery (n=3; *P<0.05 vs wild-type [WT] or nontransgenic (NTG)/sham; #P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). C, Representative colocalization images of SIRPA and TLR4 in HEK293T cells. Red, TLR4; green, SIRPA; blue, nucleus. D, SIRPA interacts with TLR4. Western blot with the Flag or Myc antibody after the coimmunoprecipitation of TLR4 from HEK293T whole-cell lysates using the Flag antibody (left). Western blot with the Flag or Myc antibody after the coimmunoprecipitation of SIRPA from HEK293T whole-cell lysates using the Myc antibody (right). E and F, Phosphorylation and total protein levels of IκBα and P65 in samples from (E) WT and SIRPA knockout mice and (F) NTG and SIRPA transgenic mice 2 weeks after AB surgery (n=3; *P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing, Knock-Out, Transgenic Assay, Western Blot, Phospho-proteomics

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Disruption of Toll-like receptor 4 (TLR4) rescued the adverse effect of signal regulatory protein-α (SIRPA) deficiency on cardiac hypertrophy. A, Images of heart sections stained with H&E and WGA from the indicated groups (n=5 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=11 mice per experimental group). F and G, Phosphorylation and total protein levels of IκBα and P65 in samples from the indicated groups 4 weeks after aortic banding(AB) surgery (n=3 independent experiments). Representative blots (F); quantitative results (G). DKO indicates double knockout.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Disruption, Staining, Phospho-proteomics, Double Knockout