mouse ccr2 apc-conjugated antibody  (Bio-Techne corporation)

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A Flowcytometric analysis of the macrophage subsets in visceral adipose tissue of CX 3 CR1 +/GFP mice which express GFP under the CX 3 CR1promoter. B tdTomato + (CX 3 CR1 high ) macrophages were quantified at day 7 after a single i.p injection of tamoxifen in CX 3 CR 1 CreER/+ ROSA tdTomato/+ mice that express YFP under the CX 3 CR1 promoter and tdTomato in CX 3 CR1-expressing cells upon tamoxifen injection using intravital microscopy (n = 6/group). C Parabiosis between C57BL/6 and CX 3 CR1 GFP/+ mice was performed. Flow cytometry was conducted to enumerate chimerism in the macrophage subsets in the C57BL/6 mice six months after parabiosis. (n = 5 for CX 3 CR1 − and 6 for CX 3 CR1 + ). D Heatmap displaying the genes with at least a two-fold difference between the VAT macrophage subsets and with FDR < 0.01 (n = 3/group). E – G Heatmaps displaying the expression of the genes using bulk RNA sequencing comparing CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − macrophages (n = 3/group) and CD206 − and CD206 + macrophage subsets of VAT (n = 3/group). H q-PCR quantification of the genes associated with glycemia and diabetes in CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − macrophages sorted from VAT of lean mice (n = 6–12/group). I Bar graph representing the frequency of CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − VAT macrophages enriched in the insulin sensitivity and resistance genes shown in ( G , H ) (n = 3-6 /group). J PCA plot showing the relations among the genes responsible for insulin sensitivity, survival, resident macrophage (ATM) markers, inflammation, insulin resistance, and monocyte-derived macrophages (MDM) markers in the VAT macrophage subsets using bulk RNA sequencing. K , L Frequencies of CCR2 + and CCR2 − macrophage subsets in human VAT as measured by flow cytometry ( K ) (n = 5/group) and confocal microscopy ( L ) (n = 15 for lean and 13 for obese). M – O Quantification of the VAT macrophage subsets in HFD-fed mice by flow cytometry (n = 4/group) ( M ) and serial intravital microscopy (Scale bar = 10 µm) ( N , O ) was performed in lean and obese CX 3 CR1 CreER/+ ROSA tdTomato/+ mice (n = 3 for CD, 4 for HFD 2 months, and 5 for HFD 4 months). P – R Apoptosis in VAT resident and monocyte-derived macrophages in lean and obese mice was examined using annexin V by flow cytometry ( P ) (n = 4/group), and caspase 3 staining by flow cytometry ( Q ) (n = 5 for CD and 4 for HFD/group) and confocal microscopy ( R ) (n = 14/group). S Quantification of the VAT macrophage subsets in lean and obese CX 3 CR1 creER/+ ROSA tdTomato mice before and after removal of HFD (n = 7/group). T Evaluation of Ki-67 + VAT resident macrophages after HFD withdrawal (n = 10/group). Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann–Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups (Fig. 1O and S).

Article Snippet: We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 #553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4#128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8#123114), CD45.1 (BioLegend, A20 #110730), CD45.2 (BioLegend,104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261).

Techniques: Injection, Expressing, Intravital Microscopy, Flow Cytometry, RNA Sequencing, Derivative Assay, Confocal Microscopy, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A The heatmap displays the levels of the cell survival regulating genes in CX3CR1 − CCR2 − VAT resident macrophages compared to CX3CR1 + CCR2 + VAT monocyte-derived macrophages using RNA sequencing (n = 3/group). B , C SerpinB2 expression in various organs ( B ) (n = 4/group) and adipose cells ( C ) (n = 4/group) is assessed using qPCR. A – C n represent the number of mice the cells or organs were obtained from. D In PMA-differentiated THP-1 macrophages, SerpinB2 levels were measured in the cell lysates and conditioned medium by immunoblot. The proinflammatory cytokines in the conditioned media were quantified by ELISA (n = 12–16/group). E – G SerpinB2 quantification in mouse CX 3 CR 1 + CCR2 + and CX 3 CR 1 − CCR2 − VAT macrophages by qPCR (E) (n = 3/group), flow cytometry ( F ) (n = 4/group), and confocal microscopy ( G ) (n = 12/group, each dot represents one tissue section.) in lean mice. The dashed and solid arrows in ( G ) indicate CCR2 + and CCR2 − macrophages, respectively. Scale bar = 20 µm. H – J Serpin B2 expression in human CCR2 + and CCR2 − VAT macrophages were measured by qPCR ( H ) (n = 5/group, each dot represents one mouse.), confocal imaging ( I ) (n = 12/group), Scale bar = 30 µm, and flow cytometry ( J ) (n = 8/group, each dot represents one mouse). K Schematic diagram depicting the experiments performed with patient omental VAT. Created in BioRender. Dutta, P. (2025) https://BioRender.com/0h4nb79 . L , M SerpinB2-expressing macrophages in lean and obese human VAT were quantified by confocal microscopy ( L ) (n = 16/group) and flow cytometry ( M) (n = 9 for lean and 12 for obese). N Correlation between BMI and SerpinB2 + cells in human VAT using confocal imaging (n = 21). O , P The frequency of SerpinB2 + macrophages in VAT of lean and obese mice by confocal microscopy ( O ) (n = 12 for lean and 11 for obese) and flow cytometry ( P ) (n = 10/group) is discerned. Q , R SerpinB2 mRNA was measured in VAT resident macrophages of lean and obese humans ( Q ) (n = 4/group, each dot represents one human sample) and mice ( R ) (n = 5/group) by qPCR. S SerpinB2 was quantified by qPCR in BMDM after palmitate treatment (n = 7/group, each dot represents cells cultured in one well.). Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann Whitney test (two-tailed) was used to determine the significance between two groups. Linear regression analysis was performed for the data presented in ( D ).

Article Snippet: We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 #553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4#128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8#123114), CD45.1 (BioLegend, A20 #110730), CD45.2 (BioLegend,104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261).

Techniques: Derivative Assay, RNA Sequencing, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Confocal Microscopy, Imaging, Cell Culture, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A , B Cytochrome c (Cyt C) levels in the mitochondria and cytoplasm measured by immunoblot in SerpinB2 − / − or SerpinB2 +/+ BMDM (n = 6/group). C , D Antioxidant gene expression using bulk RNA sequencing ( C ) (n = 3/group) and qPCR ( D ) (n = 6/group, each dot represents one mouse.) in the VAT macrophage subsets of lean mice is shown. E The bar graph represents the frequency of the VAT macrophage subsets expressing the GSH-encoding genes shown in ( C , D ) (n = 9/group). F Antioxidant gene expression using qPCR in CX3CR1 − CCR2 − VAT macrophages isolated from obese mice (n = 6/group). G The PCA plot shows the relation between GTT and GSH gene expression in macrophages of lean and obese mice. H Heat map showing the expression of the genes responsible for transporting GSH between cytoplasm and mitochondria (n = 3/group). I Antioxidant gene expression using qPCR in total macrophages sorted from obese SerpinB2 +/+ and SerpinB2 − / − mice (n = 6/group). J , K Mitochondrial ROS was measured in untreated and palmitate-treated SerpinB2 +/+ and SerpinB2 − / − BMDM isolated from lean mice and obese mice by confocal microscopy ( J ) (n = 8/group) and flow cytometry ( K ) (n = 5/group). L , M Cytosolic and mitochondrial Cytochrome c quantification in SerpinB2 − / − BMDM by immunofluorescence microscopy ( L ) (n = 7 for the without mitotempol group and 5 for with mitotempol group, each dot represents cells cultured in one well.) and assessment of annexin V MFI in SerpinB2 − / − or SerpinB2 +/+ BMDM by flow cytometry ( M ) (n = 4 /group, each dot represents one mouse.) after mitotempol treatment. Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann–Whitney test (two-tailed) was used to determine the significance between two groups.

Article Snippet: We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 #553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4#128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8#123114), CD45.1 (BioLegend, A20 #110730), CD45.2 (BioLegend,104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261).

Techniques: Western Blot, Gene Expression, RNA Sequencing, Expressing, Isolation, Confocal Microscopy, Flow Cytometry, Immunofluorescence, Microscopy, Cell Culture, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A Oxygen consumption rate (OCR) was measured by Seahorse XF 96 Extracellular Flux Analyzer (n = 20/group) in BMDM isolated from lean and obese mice. The correlation between maximal OCR and cytokine levels measured by ELISA in BMDM supernatants is shown. B GTT, ITT, and fasting insulin levels in HFD-fed LysM +/+ COX10 fl/fl and LysM cre/+ COX10 fl/fl mice (n = 7 for WT and 8 for KO, combined data of two independent experiments). C The Seahorse traces and maximal OCR (n = 24/group) in BMDM of obese SerpinB2 − / − or SerpinB2 +/+ mice are shown. D mRNA and protein levels of pro- and anti-inflammatory cytokines by qPCR and ELISA, respectively, were determined after SerpinB2 overexpression in THP-1 macrophages (n = 4/group, each dot represents cells cultured in one well.). E GTT and fasting insulin levels (n = 6–8/group, combined data of two independent experiments) (n = 8 for WT and 9 for KO) in obese SerpinB2 +/+ and SerpinB2 − / − mice. F – L HFD-fed LysM +/+ SerpinB2 fl/fl and LysM cre/+ SerpinB2 fl/fl mice were injected with PBS-vehicle, and LysM cre/+ SerpinB2 fl/fl mice were injected with IL-4. F GTT and ITT were performed, and the concentrations of fasting blood glucose, serum insulin, triglycerides (TG), free fatty acids (FFA), and free glycerol (FG) were evaluated (n = 10 for WT+vehicle, 18 for KO+vehicle, and 11 for KO + IL-4, combined data of at least two independent experiments). G Glucose infusion rate (GIR), hepatic endogenous glucose production (EGP), plasma insulin, and FFA levels are determined by hyperinsulinemic-euglycemic clamp studies (n = 7–9/group). Immunoblots ( H , I ) show pAkt, total Akt, Glut-4, adiponectin, and Ppar y expression in muscle and VAT (n = 5-6/group). The expression of the metabolic and inflammatory genes is measured by qPCR in whole VAT ( J ) (n = 8/group) and sorted VAT macrophages ( K ) (n = 6/group). L The expression of the anti-inflammatory genes was determined by RNA sequencing in CX 3 CR 1 + CCR2 + and CX 3 CR 1 − CCR2 − VAT macrophages isolated from lean mice (n = 3/group). Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann–Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups (Fig. 6F, J).

Article Snippet: We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 #553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4#128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8#123114), CD45.1 (BioLegend, A20 #110730), CD45.2 (BioLegend,104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Over Expression, Cell Culture, Injection, Clinical Proteomics, Western Blot, Expressing, RNA Sequencing, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A , B BODIPY images, adipocyte diameters and frequencies, and the expression of VAT expansion genes are shown in obese LysM cre/+ / Chr2 fl/fl mice exposed to either white (WLE) or blue (BLE) light (n = 8/group), and obese LysM +/+ SerpinB2 fl/fl and LysM cre/+ SerpinB2 fl/fl mice treated with vehicle and LysM cre/+ SerpinB2 fl/fl mice treated with IL-4 (n = 7/group). Scale bar=20 µm for BODIPY and bright field images, and 200 µm for VAT images. C Immunoblot images of the proteins in VAT of obese LysM cre/+ / Chr2 fl/fl , LysM +/+ SerpinB2 fl/fl , and LysM cre/+ SerpinB2 fl/fl mice (n = 5–6/group). D Confocal imaging to show the association between CCR2 − macrophages and adipocyte size in mouse VAT (n = 16/group). Scale bar =20 µm. E BODIPY and bright field images using ImageStreamX Mark II and Oil Red O pictures of undifferentiated or differentiated 3T3L1 cells cultured in the presence or absence of IL-4 or NAC (n = 7/group). Scale bar = 5 µm for the ImageStreamX images and 200 µm for the Oil Red O pictures. F The metabolic and inflammatory genes were measured by qPCR in 3T3 L1 differentiated adipocytes exposed to vehicle or NAC (n = 8/group). G – J Obese LysM cre/+ SerpinB2 fl/fl mice were supplemented with either water or NAC-containing water. G GTT and ITT were performed, and the concentrations of fasting blood glucose, serum insulin, triglycerides (TG), free fatty acids (FFA), and free glycerol (FG) were evaluated. (n = 8/group). H Immunoblots showing pAkt, total Akt, Glut4, adiponectin, and Ppar y expression in muscle and VAT (n = 6/group). I The expression of the metabolic and inflammatory genes was analyzed in VAT by qPCR. (n = 12/group). J Quantification of CCR2 − macrophages in VAT. (n = 14/group). I , J Each dot represents one mouse. Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups (Fig. 7B). The Mann–Whitney test (two-tailed) was used to determine the significance between two groups.

Article Snippet: We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 #553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4#128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8#123114), CD45.1 (BioLegend, A20 #110730), CD45.2 (BioLegend,104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261).

Techniques: Expressing, Western Blot, Imaging, Cell Culture, MANN-WHITNEY, Two Tailed Test