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Flow sorting from organ material by intracellular markers
Author(s) -
Moerch Ulrik,
Nielsen Henriette S.,
Lundsgaard Dorthe,
Oleksiewicz Martin B.
Publication year - 2007
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20418
Subject(s) - cell sorting , intracellular , flow cytometry , calcitonin , thyroglobulin , biology , microbiology and biotechnology , immunostaining , western blot , thyroid , gene , biochemistry , immunology , endocrinology , immunohistochemistry
Background: Fluorescence‐activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation. Methods and Results: Using the rat thyroid gland as model, rare (parafollicular) and frequent (follicular) endocrine cell types were sorted based on immunostaining for intracellular calcitonin peptide and thyroglobulin protein expression. The sorted cells were compatible with Western blot analysis of proteins, immunoassay detection of calcitonin peptide hormone and RT‐PCR. Conclusion: We developed a robust FACS protocol that allows flow sorting of rare cells from dissociated organ material, based on intracellular markers. Our FACS protocol is compatible with downstream analysis of proteins, peptides, and mRNA in the sorted cells. © 2007 International Society for Analytical Cytology