Journal: PLoS ONE

Article Title: C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehog in the mouse brain

doi: 10.1371/journal.pone.0229362

Figure Lengend Snippet: (A-B) Immunostaining of coronal brain sections from an adult mouse of the cerebral cortex and corpus callosum with the C9C5 antibody (yellow) and the oligodendroglial markers (A) CC1 (green) and Sox10 (red) or (B) CAII (green) and PDGFRα (red). White boxes (A, B) highlight magnifications of the cerebral cortex and the corpus callosum. (A) C9C5/CC1/Sox10 triple positive cells (white arrowhead) with stellate morphology are presented in merge and single channels with the nuclear marker DAPI. (B) C9C5 + (white arrowhead), CAII + (orange arrow), and PDGFRα + (white arrow) cells are presented in merge and single channels with the nuclear marker DAPI. Note that C9C5 positive cells are CAII and PDGFRα negative. Staining was replicated at least on three mice. Ctx, cerebral cortex; cc, corpus callosum.

Article Snippet: The primary antibodies were incubated overnight at 4°C: rabbit anti-SHHN (1/300, C9C5, #2207, Cell Signaling), mouse anti-GFAP (1/400, MAB360, Millipore), goat anti-Olig2 (1/400, AF2418, R&D Systems), mouse anti-S100β (1/500, S2532, Sigma), mouse anti-adenomatous polyposis coli (APC) (1/600, clone CC1, OP80, Millipore), chicken anti-βgalactosidase (1/200, ab9361, Abcam), goat anti-Sox10 (1/100, AF2864-SP, R&D Systems), rat anti-PDGFRα (1/300, 558774, BD Pharmingen), sheep anti-Carbonic Anhydrase II (CAII) (1/200, AHP206, BIORAD), rabbit anti-Iba1 (1/500, 019–19741, Fujifilm Wako), mouse anti-HuC/D (1/400, A-21271, Molecular Probes).

Techniques: Immunostaining, Marker, Negative Staining

Journal: PLoS ONE

Article Title: C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehog in the mouse brain

doi: 10.1371/journal.pone.0229362

Figure Lengend Snippet: Quantification of C9C5 + cells in oligodendrocyte populations in the adult mouse brain.

Article Snippet: The primary antibodies were incubated overnight at 4°C: rabbit anti-SHHN (1/300, C9C5, #2207, Cell Signaling), mouse anti-GFAP (1/400, MAB360, Millipore), goat anti-Olig2 (1/400, AF2418, R&D Systems), mouse anti-S100β (1/500, S2532, Sigma), mouse anti-adenomatous polyposis coli (APC) (1/600, clone CC1, OP80, Millipore), chicken anti-βgalactosidase (1/200, ab9361, Abcam), goat anti-Sox10 (1/100, AF2864-SP, R&D Systems), rat anti-PDGFRα (1/300, 558774, BD Pharmingen), sheep anti-Carbonic Anhydrase II (CAII) (1/200, AHP206, BIORAD), rabbit anti-Iba1 (1/500, 019–19741, Fujifilm Wako), mouse anti-HuC/D (1/400, A-21271, Molecular Probes).

Techniques: Marker

Journal: Nature immunology

Article Title: Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly

doi: 10.1038/s41590-020-0646-0

Figure Lengend Snippet: List of antibodies used in flow cytometry.

Article Snippet: Stains were performed for 30 minutes at 4 o C and followed by two washes with PBS containing 2% FCS and 2 mM EDTA before fixation in 0.5% PFA in PBS. table ft1 table-wrap mode="anchored" t5 Table 2.2: caption a7 Name Supplier Type Clone CD3 Biolegend Mouse IgG2a HIT3a CD11b Biolegend Mouse IgG1 ICRF44 CD14 Biolegend Mouse IgG2a M5E2 CD14 Biolegend Mouse IgG1 HCD14 CD16 Biolegend Mouse IgG1 3G8 CD19 Biolegend Mouse IgG1 HIB19 CD36 BD Biosciences Mouse IgM CB38 (NL07) CD51 (ITGAV) Miltenyi Biotec Recombinant IgG1 REA181 CD56 Biolegend Mouse IgG2a MEM-188 CD62L Biolegend Mouse IgG1 DREG-56 CD66b Biolegend Mouse IgM G10F5 CD95 (Fas) Biolegend Mouse IgG1 DX2 CD120a (TNFRI) Miltenyi Biotec Recombinant IgG1 REA252 CD120b (TNFRII) Biolegend Rat IgG2a 3G7A02 CD163 Biolegend Mouse IgG1 RM3/1 CD181 (CXCR1) Biolegend Mouse IgG2b 8F1/CXCR1 CD182 (CXCR2) Biolegend Mouse IgG1 5E8/CXCR2 CD192 (CCR2) BD Biosciences Mouse IgG2a LS132.1D9 (1D9) CD197 (CCR7) Biolegend Mouse IgG2a G043H7 CD206 Biolegend Mouse IgG1 15-2 Siglec-8 Biolegend Mouse IgG1 837535 HLA-DR BD Biosciences Mouse IgG2a G46-6 MerTK eBioscience Rat IgG2a DS5MMER TIM-4 Biolegend Mouse IgG1 9F4 P-p38 eBioscience Mouse IgG2b 4NIT4KK p65 (RelA) Biolegend Mouse IgG2b 14G10A21 P-STAT-3 (Tyr705) Biolegend Mouse IgG1 13A3-1 Live/Dead Zombie NIR Biolegend N/A N/A Open in a separate window List of antibodies used in flow cytometry.

Techniques: Cytometry, Recombinant

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Elevated neutrophil counts in the AnxA1 −/− BM. WT and AnxA1 −/− mice were sacrificed, and BM cells were extracted. Differential cell counts were conducted using Turks solution. A ) BM neutrophil counts in the BM of WT and AnxA1 −/− mice. Results are means ± se of 5 animals/group. * P < 0.05 vs . WT group. B ) Granulopoiesis from WT and AnxA1 −/− mice was assessed ex vivo . BM cells were cultured in semisolid medium, and GEMM, GM, G, and M CFUs were scored at d 12. C ) WT and AnxA1 −/− mice were injected with vehicle or G-CSF (100 μg/kg) i.p. daily for 4 d. BM was harvested 24 h after the last injection, and neutrophil levels were quantified. Neutrophil counts in the BM were assessed by flow cytometry (Ly6G high cells). Data represent means ± se of 4–11 animals/group. * P < 0.05 vs . WT group; # P < 0.05 vs . respective vehicle group.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: Ex Vivo, Cell Culture, Injection, Flow Cytometry

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Elevated levels of senescent neutrophils in the AnxA1 −/− BM. BM cells from WT and AnxA1 −/− cells were isolated. A ) Chemotatic response to various concentrations of CXCL1 was assessed for purified BM neutrophil populations, derived from both WT and AnxA1 −/− animals. B ) Expression level of CXCR2 was determined on Ly6G high cells in the BM of WT and AnxA1 −/− mice by flow cytometry. C ) Total number of apoptotic neutrophils was evaluated as the total number of cells staining positive for anxnexin V +ve /Ly6G high in the cell suspensions isolated from BM of WT and AnxA1 −/− mice. Results are means ± se of 5 animals per group. * P < 0.05 vs . WT vehicle group; # P < 0.05.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: Isolation, Purification, Derivative Assay, Expressing, Flow Cytometry, Staining

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Elevated expression of CXCR4 on BM neutrophils from AnxA1 −/− mice. BM cells from WT and AnxA1 −/− cells were isolated. A , B ) Proportion of CXCR4 expressing Ly6G high cells ( A ) and levels of CXCR4 in Ly6G high cells ( B ) in these isolated cells were determined following immunostaining. C ) Chemotatic response to various concentrations of CXCL12 was assessed for purified BM neutrophil populations from both WT and AnxA1 −/− animals. D ) Chemotatic response to various concentrations of CXCL12 was assessed for purified BM neutrophil populations from both WT and AnxA1 −/− animals that had been depleted of annexin V +ve cells. E ) Increased lipofuscin levels in AnxA1 −/− BM neutrophils as assessed flow cytometry. Results are means ± se of 5 animals/group. * P < 0.05 vs . WT vehicle group; # P < 0.05.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: Expressing, Isolation, Immunostaining, Purification, Flow Cytometry

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Altered phagocytosis of human senescent neutrophils by BM, but not peritoneal, AnxA1 −/− macrophages in vitro . BM macrophages were obtained following 5 d culture of BM cells obtained from WT and AnxA1 −/− mice in GM-CSF-rich medium. Peritoneal macrophages were prepared following peritoneal lavage of WT and AnxA1 −/− mice. A , B ) Human neutrophils were obtained from healthy volunteers; after an overnight culture they were incubated with BM ( A ) or peritoneal macrophages ( B ) for 30 min. C , D ) Subsequently, MPO staining was conducted to determine the extent of neutrophil phagocytosis. Total ( C ) and cell surface-associated ( D ) AnxA1 levels were determined in both resident BM and peritoneal macrophages; values are normalized to respective isotype control. Results are means ± se of 4 animal cell preparations/group. * P < 0.05 vs . respective control group.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: In Vitro, Incubation, Staining, Control

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Normal numbers of macrophages and monocytes in the BM of AnxA1 −/− mice. BM was harvested from WT and AnxA1 −/− mice. Numbers of monocyte and macrophages were assessed by flow cytometry (F4/80 and CD115 cells). Data represent means ± se of 4 animals/group.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: Flow Cytometry

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Impaired phagocytosis of senescent neutrophils by AnxA1 −/− BM macrophages. Extent of neutrophil clearance by BM macrophages was assessed by staining freshly extracted BM cells from WT and AnxA1 −/− animals against F4/80 antigen. Subsequently, the cells were fixed, permeabilized, and stained for Ly6G +ve expression. Data represent percentages of F4/80-Ly6G +ve double-positive BM cells from untreated WT and AnxA1 −/− mice. Results are means ± se of 4 animal cell preparations/group. * P < 0.05 vs . WT group.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

doi: 10.1096/fj.11-182089

Figure Lengend Snippet: Impaired BM phagocytosis of mouse neutrophils following in vivo administration. A ) Senescent neutrophils prepared from the BM of WT and AnxA1 −/− mice were loaded with fluorescent LX beads, and 5 × 10 6 cells were injected i.v. into recipient WT or AnxA1 −/− animals. After 24 h, cytospins were prepared from freshly isolated BM. Extent of BM macrophages (Mφ) containing LX beads was determined following counterstaining with hematoxilyn; 200 LX +ve cells/animal were counted. B ) Cultured BM-derived macrophages were observed to show the same defect in phagocytosis as in vivo resident BM macrophages. This phenotype could be rescued by addition of 10 nM hrAnxA1 to the cell cultures. C ) Number of neutrophils engulfed per macrophage was assessed my measuring mean fluorescence intensity (MFI) per cell. Results are means ± se of 5 animals/group. * P < 0.05 vs . respective WT group; ** P < 0.05; # P < 0.05.

Article Snippet: In a distinct set of experiments following neutrophil isolation, the apoptotic cells were removed from both WT and AnxA1 −/− mice, using Annexin V +ve microbeads (Miltenyi Biotec Ltd. Surry, UK) per the manufacturer's instructions.

Techniques: In Vivo, Injection, Isolation, Cell Culture, Derivative Assay, Fluorescence

Journal: Blood

Article Title: Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

doi: 10.1182/blood-2011-06-362186

Figure Lengend Snippet: Figure 1. PS exposure of NB4 and APL cells. Cells were costained with PI and either FITC–annexin V or FITC-lactadherin. (A) The cells were incubated with indicated concentrations of DNR for 24 hours. Percentage of annexin V/lactadherin- binding cells was analyzed by flow cytometry. After treatment with 1M DNR, 75% of NB4 cells and 80% of APL cells were positive for lactadherin. (B) The plasma membrane of APL cells displayed green fluorescence when stained by FITC– annexin V (left) or FITC-lactadherin (right). Cell nuclei displayed red fluorescence when labeled by PI. Co-stained areas appeared yellow. Scale bars represent 10 m.

Article Snippet: Goat anti–human annexin II IgG and goat anti–human IgG were from AbD Serotec.

Techniques: Incubation, Binding Assay, Cytometry, Clinical Proteomics, Membrane, Staining, Labeling

Journal: Blood

Article Title: Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

doi: 10.1182/blood-2011-06-362186

Figure Lengend Snippet: Figure 4. Effect of lactadherin on phagocytosis of target APL cells. A total of 1 106 CMFDA-labeled target APL cells were preincubated with 2nM lactadherin or annexin V for 10 minutes at room temperature, followed by removal of free protein- containing supernatant fluid. These cells were added to 5 105 CMTPX-stained THP-1–derived Ms or HUVECs that were seeded in 12-well culture plates before analyses by flow cytometry. Phagocytosis was quantified by measuring the percent- age of CMFDA (green)–positive red fluorescence (CMTPX) phagocytes. (A) Phago- cytic index was calculated in the absence or presence of lactadherin at indicated times before 2 hours. Lactadherin enhanced the extent of phagocytosis in a time-dependent manner. Asterisk and pound sign (*, #) indicate P .05 from phagocytosis by Ms and ECs without lactadherin, respectively. (B) Phagocytic percentage of 2 hour-incubation was assayed after pretreatment of target APL cells with lactadherin and annexin V separately. Lactadherin enhanced phagocytosis, whereas annexin V decreased engulfment. *P .05.

Article Snippet: Goat anti–human annexin II IgG and goat anti–human IgG were from AbD Serotec.

Techniques: Labeling, Staining, Derivative Assay, Cytometry, Incubation

Journal: Blood

Article Title: Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

doi: 10.1182/blood-2011-06-362186

Figure Lengend Snippet: Figure 6. Effect of lactadherin on PCA of coincu- bated target APL cells and phagocytes. Target APL cells were preincubated with 2nM lactadherin or an- nexin V for 10 minutes at room temperature. Clotting time (A), intrinsic FXa (B), extrinsic FXa (C), and throm- bin (D) of 1 106 target APL cells, or 1 106 target APL cells opsonized by annexin V or lactadherin, or incuba- tion of 1 106 target cells with phagocytes (THP-1– derived Ms or HUVECs) for 2 hours, or incubation of 1 106 annexin V–opsonized or lactadherin-opsonized target cells with phagocytes for 2 hours were deter- mined. Lactadherin and phagocytes cooperatively in- creased coagulation time and reduced enzyme com- plexes of target APL cells. *P .05 compared with single target APL cells. P .05; #P .01; and **P .001 compared with the mixture ofAPL targets and phagocytes (Ms and ECs separately).

Article Snippet: Goat anti–human annexin II IgG and goat anti–human IgG were from AbD Serotec.

Techniques: Coagulation, Derivative Assay, Incubation

Journal: Blood

Article Title: Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

doi: 10.1182/blood-2011-06-362186

Figure Lengend Snippet: Figure 7. Effect of phagocytosis on plasmin formation and annexin II expression. (A) Plasmin generation of 1 106 target APL cells, 5 105 phagocytes (THP-1–derived Ms or HUVECs), or incubation of 1 106 target APL cells with 5 105 phagocytes was evaluated at the given times. Plasmin production of the coincubated cells was time dependently reduced. *P .05 compared with 0 hour time point of each group. (B) Plasmin formation of 1 106 target APL cells, with or without 2nM annexin V or lactadherin, and with or without incubation with 5 105 phagocytes (THP-1–derived Ms or HUVECs) after 2 hours was measured. Plasmin formation of 1 106 viableAPL cells is also shown. *P .05 compared with the mixture of target APL cells and phagocytes (Ms and ECs separately). (C) NB4 cells were first labeled with goat anti–human annexin II IgG, and then with an Alexa Fluor 488–conjugated secondary Ab. Annexin II expression on permeabilized untreated NB4 cells (left) and 1M DNR-treated NB4 cells (right) was viewed with confocal microscopy. The cell nuclei were counterlabeled with PI (red), and scale bar represents 10 m. (D) Nonpermeabilized or permeabilized APL cells with and without 1M DNR treatment were stained as in panel C and analyzed by flow cytometry. Annexin II expression of DNR-treated cells decreased compared with untreated viable APL cells. (E) Flow cytometry was used to quantitate annexin II expression on cells that were treated as in panel C. Cells stained with goat anti–human IgG and Alexa Fluor 488–conjugated secondary Ab were used as control (black). The percentage of annexin II–positive viable APL cells (green) and target APL cells (pink) from one patient with permeabilization were 98.3% and 35.1%, respectively (left). Middle panel showed that, compared with controls, annexin II was expressed on the surface of nonpermeabilized THP-1–derived Ms (pink) and more so on permeabilized cells (green). Permeabilized HUVECs showed an increase in annexin II (green), but, compared with controls, nonpermeabilized HUVECs (pink) showed no increase in annexin II (right).

Article Snippet: Goat anti–human annexin II IgG and goat anti–human IgG were from AbD Serotec.

Techniques: Expressing, Derivative Assay, Incubation, Labeling, Confocal Microscopy, Staining, Cytometry, Flow Cytometry, Control