Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells.
OBJECTIVE
To develop a method of labeling and micro-dissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM).


METHODS
Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model.


RESULTS
mRNA extracted from the microdissected cell population displayed marked increases in colony-stimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury.


CONCLUSIONS
The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.
Department of Medicine, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, 55 Claverick Street, Providence, RI 02903, USA.
Stephan
S
Gehring
Edmond
E
Sabo
Maryann E
ME
San Martin
Elizabeth M
EM
Dickson
Chao-Wen
CW
Cheng
Stephen H
SH
Gregory
D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D002244:Carbon / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D007728:Kupffer Cells / Q000166:cytology / Q000502:physiology; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; C072264:Kupffer cell receptor; D012333:RNA, Messenger; D011971:Receptors, Immunologic; C028433:chinese ink; D002244:Carbon; D016186:Receptor, Macrophage Colony-Stimulating Factor