Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Deriving Human Intestinal Organoids with Functional Tissue-Resident Macrophages All From Pluripotent Stem Cells.

doi: 10.1016/j.jcmgh.2024.101444

Figure Lengend Snippet: Figure 2. Direct differentiation of monocytes/macrophages from hPSCs. (A, B) Schematic and representative images of the directed differentiation of hPSCs into HPCs followed by subsequent differentiation into monocytes/macrophages (mono/ macro) using M-CSF and GM-CSF. (C) Representative flow cytometric analysis of hPSC-derived myeloid cells stained with viability 7AAD, panhematopoietic CD45, mono/macro CD14 and CD11c, and macrophage CD163. No stain controls show gating strategy. (D, E) Schematic and gross images of Day 21 mono/macro embedded in Matrigel and cultured in HIO media with and without supplemented M-CSF and GM-CSF. (F) Representative flow cytometry of Day 35 mono/macro with and without supplemented M-CSF and GM-CSF. Adv. DMEM, Advanced DMEM; CSF, colony-stimulating factor; EGF, epidermal growth factor; L-Glut, L-glutamine; mono/macro, monocyte and macrophage; P/S, penicillin /streptomycin.

Article Snippet: 31 December 2024 12:12 pm ce CJ 44 Table 1.--- Q9 Antibody Company/catalog Mouse anti-CD34 (QBEnd/10) [1:250] Sigma-Aldrich (134M-14) Rabbit anti-CD163 [1:1,500] Sigma-Aldrich (HPA046404) Goat anti-E-Cadherin (CDH1) [1:500] R&D Systems (Cat#AF648) Rat anti-F4/80 (CI: A3-1) R&D Systems (MCA497RT) Rabbit anti-Ki67 [1:200] Cell Marque (Sigma) SP6 (275R-1) DAPI [1:10,000] Sigma-Aldrich (D9542) Donkey anti-Goat Alexa Fluor 488 [1:500] Thermo Scientific (Cat#A11055) Donkey anti-Goat Alexa Fluor 568 [1:500] Thermo Scientific (Cat#A11057) Donkey anti-Mouse Alexa Fluor 488 [1:500] Thermo Scientific (Cat#A21202) Donkey anti-Mouse Alexa Fluor 546 [1:500] Thermo Scientific (Cat#A10036) Donkey anti-Mouse Alexa Fluor 647 [1:500] Thermo Scientific (Cat#A31571) Donkey anti-Rabbit Alexa Fluor 488 [1:500] Thermo Scientific (Cat#A21206) Donkey anti-Rabbit Alexa Fluor 546 [1:500] Thermo Scientific (Cat#A10040) Donkey anti-Rabbit Alexa Fluor 647 [1:500] Thermo Scientific (Cat#A31573) Donkey anti-Rat Alexa Fluor 488 [1:500] Thermo Scientific (Cat#A21208) Mouse anti-human CD11c PE Cy7 BD Pharmingen (561356) Mouse anti-human CD14 v500 BD Pharmingen (562693) Anti-human CD45 FITC Tonbo Biosciences (35-9459- T025) Anti-human CD163 APC eBioscience (17-1639-41) 7-Aminoactinomycin D (7-AAD) Thermo Scientific (A1310) 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710 1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 Alternatively, the HIOs were plated in Matrigel and the monocytes/macrophages were resuspended and added in the overlaying media.

Techniques: Derivative Assay, Staining, Cell Culture, Cytometry

Journal: PLoS ONE

Article Title: Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

doi: 10.1371/journal.pone.0058560

Figure Lengend Snippet: A, Wild type, STXBP1 mutant, or heterozygous mice were identified by PCR using DNA from liver cells. B , After 4–5 weeks of culture, liver-derived mast cells (LMC) were examined by toluidine blue staining to determine mast cell purity. A representative photomicrograph is shown (original magnification = ×100). C , LMCs were sensitized with anti-TNP IgE, stained with FITC-conjugated anti-IgE or anti-c-kit antibodies, and examined by flow cytometry. D, STXBP1 +/+ and STXBP1 −/− LMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA (10 ng/ml) for various times. Whole cell lysates were analyzed by Western blotting for STXBP1 and Actin. E, Sec1/Munc18 (SM) family gene expression profiles were identified by RT-PCR using mRNA from wild type mouse bone marrow-derived mast cells (BMMC), and wild type (STXBP1 +/+ ) or STXBP1 −/− LMCs, with or without TNP-BSA stimulation. All members of SM family are expressed in wild type BMMCs and LMCs. Similar results were observed in 2 independent experiments.

Article Snippet: FITC-conjugated rat anti-mouse IgE (IgG1), FITC-rat IgG1 and FITC-conjugated rat anti-mouse CD117 (c-kit) were purchased from Cedarlane Laboratories (Burlington, ON, Canada).

Techniques: Mutagenesis, Derivative Assay, Staining, Flow Cytometry, Western Blot, Gene Expression, Reverse Transcription Polymerase Chain Reaction