C57Bl/6 and FSTL-1 Hypomorphic (on C57Bl/6 background) mice were housed in accordance with the approved University of Pittsburgh School of Medicine Institutional Animal Care and Use Committee protocol12 (link). The bone marrow compartment of male and female wild-type and FSTL-1 Hypomorhic mice was isolated by femur flush protocol50 . Isolated bone marrow cells were resuspended in sterile phosphate-buffered saline and counted using trypan blue stain and a hemocytometer. The cells were first blocked using anti-mouse CD16/CD32 (eBioscience) and subsequently surfaced stained in the presence of anti-mouse CD45 antibody (clone 30-F11, BD Bioscience). CD45-positive and CD45-negative cell populations were then sorted on a FACSAria via FACSDiva software (Becton-Dickinson) into phosphate-buffered saline. The sorted cell populations were pelleted and resuspended in RNA Lysis Buffer (Zymo Research). RNA was processed for gene expression analysis as previously described. These experiments were performed twice with n=3 or n=4 per group each time. Data shown include cells from all animals in the two experiments.
Anti mouse cd45 antibody clone 30 f11
Manufactured by BD
The Anti-mouse CD45 antibody (clone 30-F11) is a lab equipment product used for the identification and characterization of mouse CD45-expressing cells. It is a purified monoclonal antibody that binds specifically to the CD45 cell surface antigen found on various hematopoietic cells in mice.
Automatically generated - may contain errors
3 protocols using anti mouse cd45 antibody clone 30 f11
1
Isolation and Analysis of Murine Bone Marrow
2
Isolation and Analysis of Murine Bone Marrow
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C57Bl/6 and FSTL-1 Hypomorphic (on C57Bl/6 background) mice were housed in accordance with the approved University of Pittsburgh School of Medicine Institutional Animal Care and Use Committee protocol12 (link). The bone marrow compartment of male and female wild-type and FSTL-1 Hypomorhic mice was isolated by femur flush protocol50 . Isolated bone marrow cells were resuspended in sterile phosphate-buffered saline and counted using trypan blue stain and a hemocytometer. The cells were first blocked using anti-mouse CD16/CD32 (eBioscience) and subsequently surfaced stained in the presence of anti-mouse CD45 antibody (clone 30-F11, BD Bioscience). CD45-positive and CD45-negative cell populations were then sorted on a FACSAria via FACSDiva software (Becton-Dickinson) into phosphate-buffered saline. The sorted cell populations were pelleted and resuspended in RNA Lysis Buffer (Zymo Research). RNA was processed for gene expression analysis as previously described. These experiments were performed twice with n=3 or n=4 per group each time. Data shown include cells from all animals in the two experiments.
3
Liver Inflammation Markers and Immune Cell Profiling
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The histopathological (hematoxylin and eosin (H&E) staining) and level of liver transaminases (ALT/AST) in serum were performed as described earlier. Immunolocalisation of IL-33 was performed by immunohistochemical staining using primary antibody anti-mouse-IL-33 (goat IgG, R&D Systems). Leucocytes were stained with anti-mouse CD45 antibody (clone 30-F11, BD Pharmingen), macrophages with anti-mouse F4/80 antibody (clone BM8, eBioscience), and neutrophils, after antigen retrieval step (citrate buffer pH 6), with anti-mouse Ly6G antibody (clone 1A8, BD Bioscience). Then, secondary HRP-conjugated dedicated specific antibodies (Dako, USA) were used followed by hematoxylin counterstaining in a Ventana machine (Ventana Medical Systems Inc., USA). The counting of stained positive cells was carried in at least 3 different microscopic fields of 1 mm2 surface area by tissues on at least 3 different tissues by conditions by using image analysis software (NDP, view, Hamamatsu).
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