fitc anti human itga6  (Miltenyi Biotec)

Journal: Clinical Science (London, England : 1979)

Article Title: Protective effect of mesenchymal stromal cells in diabetic nephropathy: the In vitro and In vivo role of the M-Sec-tunneling nanotubes

doi: 10.1042/CS20242064

Figure Lengend Snippet: (A) The diagram shows the experimental design. MSCs were isolated from the bone marrow of non-diabetic (MSC-ND) and diabetic (MSC-DM) mice and then incubated with podocytes pre-exposed to advanced glycosylated end products-bovine serum albumin (AGEs)/vehicle. In selected experiments, MSC-DM were transfected with an adenovirus to overexpress Miro1 or a mock vector. (B) Isolated MSCs were positive for CD44, CD49f, SCA-1, and negative for CD45 as assessed by flow cytometry. (C) MSC were cultured in Mesencult Adipogenic Differentiation Medium for 7 days. Oil Red O staining revealed the presence of fat droplets in the cytosol, proving MSC differentiation into adipocytes. Magnification X400, scale bar 50 µm. (D) Podocytes labelled with CellTracker Blue were pre-exposed to AGEs/vehicle and then co-cultured with MSC-ND and MSC-DM cells. Cells were stained with Alexa Fluor 488-WGA to reveal TNTs and the percentage of podocytes connected with MSCs via TNTs counted (n=3 *** P <0.001 AGEs-MSC-ND and AGEs-MSC-DM vs. vehicle-MSC-ND). (E) Podocytes labelled with CellTracker Blue were pre-exposed to AGEs/vehicle and then co-cultured with MSC-ND and MSC-DM, carrying RFP-labelled mitochondria. The percentage of blue podocytes containing MSC-derived RFP-mitochondria (dually labelled podocytes) was quantified by flow cytometry (n=3 *** P <0.001 AGEs-MSC-ND vs. AGEs-MSC-DM and vehicle-MSC-ND). (F,G) Miro1 mRNA and protein expression was assessed in MSC-ND and MSC-DM cells by immunoblotting (internal control: tubulin) (n=3, ** P <0.01 MSC-DM vs. MSC-ND) and real-time PCR (housekeeping gene: GAPDH ) (n=3 ** P <0.01 MSC-DM vs. MSC-ND), respectively. (H) Miro1 mRNA levels were measured in MSC-ND exposed to vehicle, AGEs, and MCP1 for 24 h (n=3 *** P <0.01 MCP1 vs. vehicle, ** P <0.01 AGEs vs. vehicle) (I) MSC-DM were transfected with an adenovirus to overexpress Miro1 (MSC-DM-Miro) or a mock vector (MSC-DM-Mock) and efficiency of transfection assessed by measuring Miro1 mRNA by real-time PCR (n=3 ** P <0.01 DM-Mock vs. ND and DM-Miro1). (J) Podocytes labelled with CellTracker Blue were pre-exposed to AGEs/vehicle and then co-cultured with MSC-ND, MSC-DM-Miro or MSC-DM-Mock, carrying RFP-labelled mitochondria. The percentage of blue podocytes containing MSC-derived RFP-mitochondria (dually labelled podocytes) was quantified by flow cytometry (n=3 *** P <0.01 DM-Mock vs. ND and DM-Miro1).

Article Snippet: Cells were subcultured until passage three and then characterised by flow cytometry using the following markers: FITC-CD44, FITC-SCA-1, FITC-CD49f, PE-CD45 (Thermo Fisher Scientific).

Techniques: Isolation, Incubation, Transfection, Plasmid Preparation, Flow Cytometry, Cell Culture, Staining, Derivative Assay, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: DDR1 regulates RUNX1-CBFβ to control breast stem cell differentiation

doi: 10.1101/2024.02.21.581255

Figure Lengend Snippet: A) Schematic of the strategy to test the effects of DDR1i on breast TDLU organogenesis. Inhibition during induction began with DDR1i treatment starting at day 0 and concluded when control structures were fully formed no later than day 28. Inhibition during patterning started with DDR1i beginning day 7 and concluding around day 28. B) Quantification of the types of organoids that formed following DDR1i treatment during induction. Data presented as Mean ± SD (n= 4 gels/primary patient samples). C) Representative immunofluorescence staining of organoids from control or DDR1 inhibitor-treated gels, with treatment initiated during the induction phase of organoid formation. CK14 (green), and E-Cadherin (orange), and DAPI (blue) staining. Scale bar = 200 μm. D) Quantification of the types of organoids formed in DDR1i treated cultures beginning during patterning. Data presented as Mean ± SD (n= 4 gels/primary patient samples). E) Representative immunofluorescence staining of organoids from control or DDR1i treated gels initiated during the patterning phase of organoid formation. CK14 (green), and E-Cadherin (orange), DAPI (blue) staining. Scale bar = 200 μm. F) Representative flow cytometry plot analysis of basal (CD49f) and luminal (Epcam) cells (FACS) (i) and quantification of mean fluorescent intensity (ii) from primary patient samples cultured in 3D, treated with DDR1i during patterning (n=3 primary samples, and values are expressed as Mean ± SD). Statistical significance was determined via multiple t-tests, with significance levels indicated as follows: *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001.

Article Snippet: Samples were washed and stained with the antibodies CD49f-FITC (555736, BD Biosciences, GoH3, 1:20) and EPCAM-PE (347198, BD Biosciences, 1:20).

Techniques: Inhibition, Immunofluorescence, Staining, Flow Cytometry, Cell Culture

Journal: bioRxiv

Article Title: DDR1 regulates RUNX1-CBFβ to control breast stem cell differentiation

doi: 10.1101/2024.02.21.581255

Figure Lengend Snippet: A) Quantification of the types of organoids that formed following RUNXi treatment during induction. Data presented as Mean ± SD (n= 4 gels/primary patient samples). B) Representative immunofluorescence staining of organoids from control or RUNX inhibitor-treated gels, with treatment initiated during the induction phase of organoid formation. CK14 (green), and E-Cadherin (orange), and DAPI (blue) staining. Scale bar = 200 μm. C) Quantification of the types of organoids that formed following RUNXi treatment during patterning (n= 4 gels/primary patient samples). Data presented as Mean ± SD. D) Representative immunofluorescence staining of organoids from control or RUNX inhibitor-treated gels, with treatment initiated during the induction phase of organoid formation. CK14 (green), and E-Cadherin (orange), and DAPI (blue) staining. Scale bar = 200 μm. E) Representative flow cytometry plot analysis of basal (CD49f) and luminal (Epcam) cells (FACS) (i) and quantification of mean fluorescent intensity (ii) from primary patient samples cultured in 3D, treated with RUNX inhibitor during patterning (n=3 primary samples). values are expressed as Mean ± SD. Statistical significance was assessed using multiple t-tests, with significance levels indicated as follows: *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001.

Article Snippet: Samples were washed and stained with the antibodies CD49f-FITC (555736, BD Biosciences, GoH3, 1:20) and EPCAM-PE (347198, BD Biosciences, 1:20).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Cell Culture