TY - CHAP
T1 - Multi-parametric imaging of hypoxia and cell cycle in intestinal organoid culture
AU - Okkelman, Irina A.
AU - Foley, Tara
AU - Papkovsky, Dmitri B.
AU - Dmitriev, Ruslan I.
N1 - Publisher Copyright:
© Springer International Publishing AG 2017.
PY - 2017
Y1 - 2017
N2 - Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.
AB - Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.
KW - Cell cycle
KW - FLIM
KW - Intestinal organoids
KW - Live cell microscopy
KW - Oxygen
KW - Phosphorescence quenching
KW - PLIM
UR - https://www.scopus.com/pages/publications/85032569810
U2 - 10.1007/978-3-319-67358-5_6
DO - 10.1007/978-3-319-67358-5_6
M3 - Chapter
C2 - 29080132
AN - SCOPUS:85032569810
T3 - Advances in Experimental Medicine and Biology
SP - 85
EP - 103
BT - Advances in Experimental Medicine and Biology
PB - Springer New York LLC
ER -