Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet: Activation and Killing by T Cells Is Selective for iPSC-β (A) Experimental design: PBMCs co-cultured with autologous iPSC-derived cells. (B, C, and F) CD25 or CD69 expression shown as MFI. (B) PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-α (red). (n = 3 T1D and n = 1 ND donor, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (C) Donor-matched PBMCs (CD3 + gated) (n = 1 T1D, n = 3 differentiation batches per donor line) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-cardiomyocytes (orange). (D) Percentage of live iPSC-β (C-peptide + /glucagon − ) or iPSC-α (C-peptide − /glucagon + ) from iPSC-β or iPSC-α differentiations, respectively (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (F) Representative flow cytometry histograms after 48 h of co-culture. Dashed histogram represents the control (untreated target cells). (E) Percentage of apoptotic (apopxin + ) iPSC-β (CD49a + /CD26 − ) or iPSC-α (CD49a − /CD26 + ) from iPSC-β or iPSC-α differentiations (n = 1 T1D iPSC donor, n = 3 differentiation batches per donor line). (G) Unmatched PBMCs (CD3 + gated cells) co-cultured for 48 h with iPSC-α (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (H) Donor-matched PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous enriched iPSC-β or iPSC-α. Data are means ± SEMs, 2-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001; ns, non-significant. See also Figure S4 .

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Expressing, Flow Cytometry, Co-Culture Assay, Control

Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet:

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Virus, Recombinant, Software, Real-time Polymerase Chain Reaction

Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet: Panel for flow cytometry analysis of sorted populations

Article Snippet: CD26 Antibody, anti-mouse, REAfinity- PE (1:100 dilution) , Miltenyi Biotec , Cat# 130-122-775, RRID: AB_2801934.

Techniques: Flow Cytometry, Marker, Staining

Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet: Sample detail for fluorescence compensation settings

Article Snippet: CD26 Antibody, anti-mouse, REAfinity- PE (1:100 dilution) , Miltenyi Biotec , Cat# 130-122-775, RRID: AB_2801934.

Techniques: Fluorescence, Staining, Suspension

Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet: Flow cytometry gating and isolated fraction purity/viability (A) Gating strategy for cellular subsets identification. Singlet live cells were obtained by using SSC/Time, FSC/SSC and FSC/mortality dye (LD) parameters. (B) To appreciate each fraction purity, dot plot with CD45, CD31, Lin and CD26 (CAFs marker) staining were shown in CD45+ TILs, tumor cells and CAFs in total tumor suspension. (C) Proportion of CD45+ TILs, Endothelial cells, Tumor cells and CAFs in tumor cell suspension and purity of each cell subsets in isolated fractions.

Article Snippet: CD26 Antibody, anti-mouse, REAfinity- PE (1:100 dilution) , Miltenyi Biotec , Cat# 130-122-775, RRID: AB_2801934.

Techniques: Flow Cytometry, Isolation, Marker, Staining, Suspension

Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet: Phenotypic and transcriptomic analysis (A and B) Representative flow cytometry analysis of CAFs markers (CD26, FAPa, PDPN, PDGFRa, PDGFRb) on tumor cells and CAFs fractions (A). Normalized expression of each marker (B). (C) Analysis of CAFs, CD45+ TILs and tumor associated genes expression by RT-qPCR in each isolated fraction (n = 3 samples/fraction).

Article Snippet: CD26 Antibody, anti-mouse, REAfinity- PE (1:100 dilution) , Miltenyi Biotec , Cat# 130-122-775, RRID: AB_2801934.

Techniques: Flow Cytometry, Expressing, Marker, Quantitative RT-PCR, Isolation

Journal: STAR Protocols

Article Title: Simultaneous isolation of CD45 tumor-infiltrating lymphocytes, tumor cells, and associated fibroblasts from murine breast tumor model by MACS

doi: 10.1016/j.xpro.2022.101951

Figure Lengend Snippet:

Article Snippet: CD26 Antibody, anti-mouse, REAfinity- PE (1:100 dilution) , Miltenyi Biotec , Cat# 130-122-775, RRID: AB_2801934.

Techniques: Recombinant, Flow Cytometry, Staining, Isolation, Cell Isolation, Software, Real-time Polymerase Chain Reaction, Blocking Assay, Gentle