anti human osteocalcin monoclonal antibody  (Bio-Rad)

Journal: Journal of Extracellular Vesicles

Article Title: Y‐box binding protein 1 in small extracellular vesicles reduces mesenchymal stem cell differentiation to osteoblasts—implications for acute myeloid leukaemia

doi: 10.1002/jev2.12417

Figure Lengend Snippet: MV4‐11 and K562 sEVs influence BM‐MSCs differentiation. (a) Schematic workflow of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) treatment with MV4‐11 and K562 sEVs. Small EVs were incubated with BM‐MSCs with a ratio of 50:1 (sEV particle number/recipient cells). Analysis of PKH26‐labelled MV4‐11 and K562 sEVs transferred into BM‐MSCs after 24 h using (b) confocal microscopy and (c) flow cytometry. Representative confocal images showing cell nuclei (DAPI, blue), cell membrane (WGA‐Alexa488, green) and PKH26‐labelled leukaemia sEVs (PKH26, red). Scale bars‐ 10 µm. (d) MTT assay of BM‐MSCs treated with different MV4‐11 and K562 sEVs concentrations for 24 h. (e) Colony‐forming unit (CFU) assay performed on BM‐MSCs treated with leukaemia sEVs for three weeks. (f) Relative mRNA expression of various genes important for normal hematopoietic function in bone marrow microenvironment. (g) Trilineage differentiation of BM‐MSCs treated with leukaemia and healthy control sEVs. Graphs showing the influence of adipogenic (FABP4), chondrogenic (aggrecan) and osteogenic (osteocalcin) differentiation of BM‐MSCs due to the sEVs treatment. Data shown in c‐g are mean ± S.E.M. obtained from three independent experiments, and statistical significance is calculated in reference to either untreated BM‐MSCs or BM‐MSCs treated with healthy control sEVs (**** p < 0.0001, *** p < 0.001, ** p < 0.01, and ns‐ non‐significant).

Article Snippet: BM‐MSCs treated with or without leukaemia sEVs were analysed for trilineage differentiation by flow cytometry using antibodies such as mouse anti‐human FABP4 (Cat No: ab93945, dilution‐1:100, Abcam, Cambridge, UK) + goat anti‐mouse IgG H&L with Alexa488 (Cat No: ab150113, dilution‐ 1:2000, Abcam, Cambridge, UK); rabbit anti‐human aggrecan Alexa647‐conjugated polyclonal antibody (Cat No: NB100‐74350AF647, dilution: 1:100, Novus Biologicals, CO, USA) and mouse anti‐human osteocalcin PE‐conjugated monoclonal antibody (Cat No: IC1419P, dilution‐ 1:50, Biotechne, Germany).

Techniques: Derivative Assay, Incubation, Confocal Microscopy, Flow Cytometry, Membrane, MTT Assay, Colony-forming Unit Assay, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Y‐box binding protein 1 in small extracellular vesicles reduces mesenchymal stem cell differentiation to osteoblasts—implications for acute myeloid leukaemia

doi: 10.1002/jev2.12417

Figure Lengend Snippet: Osteogenic differentiation analysis of BM‐MSCs. Small EVs were incubated with BM‐MSCs in the ratio of 50:1 (50 sEV particles per cell). (a) Representative confocal images showing the osteoblastic differentiation of BM‐MSCs treated with AML and non‐AML sEVs. Cell nuclei are shown in blue (DAPI), and the osteogenesis marker (osteocalcin) is shown in green. Scale bars—10 µm. (b) Mean Fluorescence Intensity (MFI) of osteocalcin in BM‐MSCs treated with different sEVs expressing osteocalcin. For each condition, MFI average was calculated from at least four confocal images. p values were calculated using one‐way ANOVA with Dunnett's multiple comparisons (**** p < 0.0001, *** p < 0.001, * p < 0.05 and ns‐ non‐significant). (c) Alizarin red staining of BM‐MSCs treated with AML and non‐AML sEVs.

Article Snippet: BM‐MSCs treated with or without leukaemia sEVs were analysed for trilineage differentiation by flow cytometry using antibodies such as mouse anti‐human FABP4 (Cat No: ab93945, dilution‐1:100, Abcam, Cambridge, UK) + goat anti‐mouse IgG H&L with Alexa488 (Cat No: ab150113, dilution‐ 1:2000, Abcam, Cambridge, UK); rabbit anti‐human aggrecan Alexa647‐conjugated polyclonal antibody (Cat No: NB100‐74350AF647, dilution: 1:100, Novus Biologicals, CO, USA) and mouse anti‐human osteocalcin PE‐conjugated monoclonal antibody (Cat No: IC1419P, dilution‐ 1:50, Biotechne, Germany).

Techniques: Incubation, Marker, Fluorescence, Expressing, Staining