anti fcgr2b  (Novus Biologicals)

Journal: Cell Reports Medicine

Article Title: Modeling antithymocyte globulin-induced microvasculopathy using human iPSC-derived vascularized liver organoids

doi: 10.1016/j.xcrm.2025.102433

Figure Lengend Snippet: Sinusoid-like microvasculatures in human liver organoids transplanted in the mouse cranial window (A) Schematic representation of human liver bud organoids (HLBOs) transplantation under cranial window (CW) and the engraftment process. Intravital fluorescence microscopy showing the transplanted organoid observed by GFP + hepatic endoderm (hiPSC-HE) and mCherry + liver sinusoidal endothelial cell (iLSEC) progenitors derived from human iPSC. (B–F) Immunofluorescence analysis of HLBOs 46 days after CW transplantation. (B) Co-staining for the LSEC marker LYVE1 (green) and the human nuclear antigen Ku80 (red). The left panel shows a low-magnification field (scale bars, 50 μm). The right panels show high-magnification images of the mouse brain region (upper) and the HLBO region (lower) (scale bars, 20 μm). (C) Co-staining for the endothelial marker CD31 (green) and LYVE1 (red). The left panel shows a high magnification field (scale bar, 50 μm), and the right panels show single channels comparing the intensity of CD31 and LYVE1 in a smaller-caliber vessel (upper, cyan arrows) and a larger-caliber vessel (lower, yellow arrows). Scale bars, 20 μm. (D) Quantification of vessel diameter between the LYVE1 low and LYVE1 high vessels. Each dot represents an individual vessel (mean ± SD, n = 4 per group; unpaired t test). (E) Co-staining for LYVE1 (green) and CD32B (red). The left panel shows a high magnification field (scale bars, 50 μm), and the right panels show single channels comparing the intensity of LYVE1 and CD32B of a smaller-caliber vessel (upper, cyan arrows) and a larger-caliber vessel (lower, yellow arrows) (scale bars, 20 μm). (F) Co-staining of CD31 (green) and COUP-TFII (red) in both small (upper) and large CD31 + vessels (lower) within the HLBO region. Scale bars, 20 μm.

Article Snippet: For CD32B blocking experiments, iLSECs were pre-incubated for 30 min at 37°C with either a control antibody (25 μg/mL; Santa Cruz, sc-2028) or a blocking anti-CD32B antibody (5 or 25 μg/mL; NSJ Bioreagents, R34966 ) in SEDM.

Techniques: Transplantation Assay, Fluorescence, Microscopy, Derivative Assay, Immunofluorescence, Staining, Marker

Journal: Cell Reports Medicine

Article Title: Modeling antithymocyte globulin-induced microvasculopathy using human iPSC-derived vascularized liver organoids

doi: 10.1016/j.xcrm.2025.102433

Figure Lengend Snippet: iLSECs are more susceptible to ATG-induced microvascular injury than iAECs (A) Heatmap of selected endothelial lineage markers across iAECs (day 21), iLSECs (day 21), and primary human LSECs, showing Pan-EC, arterial, venous, and LSEC-specific gene expression patterns. (B) Whole-mount immunostaining of CD32B (red) and LYVE1 (gray) in day 18 HLBOs containing GFP + iAECs or iLSECs (green). Scale bars, 100 μm. (C) Intravital confocal images showing blood flow (TRITC 2,000 kDa dextran, blue) and C3 (anti-C3 antibody, red) in GFP + iAEC- or iLSEC- lined vessels (green) 1 h before and 6 h after ATG injection. Scale bars, 100 μm. (D) Quantification of the change in blood perfusion area from 1 h before to 6 h after ATG injection in iAEC- or iLSEC-lined vessels (mean ± SD, n = 4 fields from 1 mouse per group; unpaired t test). (E) Quantification of extravascular dextran leakage at 6 h post-ATG injection in iAEC- or iLSEC-lined vessels (mean ± SD, n = 4 fields from 1 mouse per group; unpaired t test). (F) Intravital confocal images of mCherry + iLSEC-lined vessels (gray) 1 h before and 6 h after ATG injection. Blood, platelets, and C3 were visualized with GFP 2,000 kDa dextran (blue), anti-CD41 antibody (green), and anti-C3 antibody (red), respectively. Cyan arrows indicate thrombus and dextran leakage in a smaller-caliber vessel (average diameter <50 μm); yellow arrows indicate a larger-caliber vessel (average diameter >50 μm). Scale bars, 50 μm. (G and H) Quantification of C3 deposition (G) and extravascular dextran leakage (H) in smaller versus larger vessels at 6 h post-ATG injection (mean ± SD, n = 4 per group; unpaired t test).

Article Snippet: For CD32B blocking experiments, iLSECs were pre-incubated for 30 min at 37°C with either a control antibody (25 μg/mL; Santa Cruz, sc-2028) or a blocking anti-CD32B antibody (5 or 25 μg/mL; NSJ Bioreagents, R34966 ) in SEDM.

Techniques: Gene Expression, Immunostaining, Injection

Journal: Cancer Research

Article Title: Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

doi: 10.1158/0008-5472.CAN-23-2135

Figure Lengend Snippet: Accumulation, FcR expression, and tumoricidal activity of tumor-infiltrating myeloid cells. A, Frequencies of indicated FcR + effectors in lung tumors analyzed by flow cytometry in a single-cell suspension obtained from digested lung tumors. B–D, Representative dot plots and cumulative flow cytometry results showing the expression of the indicated FcRs on myeloid effectors in blood and tumors. MFI, mean fluorescence intensity. E, Representative dot plots and cumulative flow cytometry results showing the expression of CD32a and CD32b molecules on the surface of gated CD14 + HLA-DR int CD206 − blood monocytes and CD14 + HLA-DR hi CD206 + TAMs. Cells stained with isotype control Abs were used to set the gates. Wilcoxon matched paired test. F and G, Representative dot plots and cumulative flow cytometry results showing the ability of blood and tumor FcR + effectors to kill PKH-67 + A431 tumor cells in the presence of cetuximab (1 μg/mL) at a 50:1 E:T ratio in a 12-hour assay. Dead A431 tumor cells were defined as TO-PRO-3 + PKH67 + cells. Summary graphs represent the total tumoricidal activity of effectors calculated as described in Materials and Methods. H and I, Representative images of GFP + A431 tumor cells cocultured with indicated blood effectors (patient LC441) and cetuximab for 48 hours in the IncuCyte Live Cell Analysis System. Scale bar, 400 μm. Representative experiment ( H ) and summary results ( I ) showing the kinetics of GFP + A431 tumor cell growth during coculturing with blood and tumor FcR + effectors at a 50:1 E:T ratio in the presence of cetuximab (1 μg/mL) and in the presence of IgA anti-EGFR Ab (1 μg/mL; J and K ) in the IncuCyte Live Cell Analysis System. Percentage of tumor cell growth inhibition/stimulation in the presence of anti-EGFR Abs was calculated at 48 hours using the formula: (FI)(A431+Ab)-FI(A431+effectors+Ab)/FI(A431+Ab) × 100%. FI, Integrated fluorescence intensity. The number of patients included in each analysis is indicated on the graphs. All data are represented as mean ± SEM. All comparisons used one-way ANOVA with Tukey multiple comparisons tests. FcR + effectors were freshly isolated for all experiments.

Article Snippet: Anti-human CD32b (FCGR2b) recombinant antibody (clone: 6G11, HPAB-0535YY) and anti-human CD32a (FCGR2a) antibody (clone: IV.3, NEUT-790CQ) were purchased from Creative Biolabs, Inc. Anti-human CD32b (FCGR2b) recombinant antibody (clone: 2B6) with modified silent Fc fragment were kindly provided by Dr. G. Georgiou.

Techniques: Expressing, Activity Assay, Flow Cytometry, Suspension, Fluorescence, Staining, Control, Cell Analysis, Inhibition, Isolation

Journal: Cancer Research

Article Title: Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

doi: 10.1158/0008-5472.CAN-23-2135

Figure Lengend Snippet: Role of TAM-mediated ADT in facilitating tumor cell escape from tAbs. A, Scheme proposing the role of TAM-mediated ADT in the downmodulation of cetuximab/EGFR complexes on the surface of opsonized tumor cells that could lead to resistance of targets to subsequent attacks by NK cells. B, Representative flow cytometry histograms showing the expression of cetuximab/EGFR complexes on the surface of cetuximab-opsonized A431 cells cocultured in the presence or absence of TAMs at a 1:1 E:T ratio for 2 hours. C, Cumulative results showing the expression of cetuximab/EGFR complexes on the surface of cetuximab-opsonized A431 and A549 cells cocultured in the presence or absence TAMs at a 1:1 E:T ratio for 2 hours. Wilcoxon matched-pairs signed rank test. D and E, Representative dot plots and cumulative flow cytometry data demonstrating the reduced ability of blood NK cells to kill cetuximab-opsonized A431 and A549 tumor cells in the presence of TAMs. Tumor cell lines were preopsonized with cetuximab and mixed with TAMs at a 1:1 E:T ratio; two hours later, NK cells were added at a 10:1 E:T ratio for additional 12 hours. Wilcoxon matched pairs signed rank test. Some experiments were performed with blocking anti-CD64 F(ab')2 and anti-CD32 F(ab')2 Abs (5 μg/mL) and representative dot plots from one of three experiments are shown. F, The kinetics of A431 tumor cell growth when cocultured with TAMs and blood NK cells in the presence of cetuximab in the IncuCyte Live Cell Analysis System. A431 cells were preopsonized with cetuximab and mixed with TAMs at a 3:1 E:T ratio; two hours later, NK cells were added at a 10:1 E:T ratio. The ability of TAMs to mediate ADT was assessed by flow cytometry in the cocultures of TAMs and cetuximab-opsonized PKH67 + A431 cells as described earlier. Two patients (LC#643 and LC#649) are shown. All data represented as mean ± SEM. FcR + effectors were freshly isolated for all experiments. ( A, Created with BioRender.com.)

Article Snippet: Anti-human CD32b (FCGR2b) recombinant antibody (clone: 6G11, HPAB-0535YY) and anti-human CD32a (FCGR2a) antibody (clone: IV.3, NEUT-790CQ) were purchased from Creative Biolabs, Inc. Anti-human CD32b (FCGR2b) recombinant antibody (clone: 2B6) with modified silent Fc fragment were kindly provided by Dr. G. Georgiou.

Techniques: Flow Cytometry, Expressing, Blocking Assay, Cell Analysis, Isolation