Flow sorting from organ material by intracellular markers.
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.


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.


CONCLUSIONS
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.
Moerch
Ulrik
U
Department of Virology and Molecular Toxicology, Novo Nordisk A/S, Maaloev, Denmark.
Nielsen
Henriette S
HS
Lundsgaard
Dorthe
D
Oleksiewicz
Martin B
MB
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D015415:Biomarkers / Q000032:analysis; D002116:Calcitonin / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013954:Thyroglobulin / Q000276:immunology / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology*
D000906:Antibodies; D015415:Biomarkers; D002116:Calcitonin; D013954:Thyroglobulin