Environmental Engineering Reference
In-Depth Information
Mono mac 6 cells or IC-21 lines or as primary cultures, e.g. alveolar macrophages.
Gastric, nasal, oral, and urinary tract epithelium are all characterized in vivo by
viscous mucin production and tissue coatings that is absent in cell-based assays,
which does not represent in vivo condition. The condition maintained in vitro must
be as close as possible to the in vivo scenario. Long et al. used BV2 cells
(immortalized mouse microglia), rat dopaminergic (DA) neurons (N27), and pri-
mary cultures of embryonic rat striatum to model the characteristic ROS-burst from
particle insult to these macrophage-like cells in the context of the brain [
14
].
The changes in cells are indication of modulation of protein or gene expression,
phagocytic ability, and/or inflammation reactions that alter normal phenotype.
Short-lived ROS species are main cause of these changes and can be determined
in cell media by fluorescein-compound-based tests (fluorescein probes,
2
0
,7
0
-difluorescein-diacetate dichlorodihydrofluorescein diacetate) or by Electron
Paramagnetic Resonance (EPR). ROS in cell may be quantified by the well-known
glutathione (GSH) assay. Other assays are based on the analysis of key oxidized
species damage cell membranes or DNA mainly by immunocytochemistry which
allows detection of specific DNA lesions and directly measures the ROS involve-
ment in DNA damage. BODIPY-C11 is a fluorescent dye which adapts different
colors when oxidized and unoxidized into lipid bilayers, is another way to deter-
mine stress or lipid peroxidation at cell membrane. Figure
6.4
numbers the cell lines
available for various infectious and non-infectious diseases according to ATCC.
Cell viability assay gives indication of live cells as a response to dose and also
answers question of biocompatibility. Various dye-based assays are used widely
which mainly employ neutral red, trypan blue. Other assays which determine cell
viability are LIVE/DEAD
kits by Life Technologies, lactate dehydrogenase
(LDH), formazan-based assays (MTT), MTS, WST, Alamar blue (resazurin),
Coomassie blue, ATP-luciferin luminescence, adenylate kinase (AK) release
assay, determination of mitochondrial membrane potential (MMP) and
thiobarbituric (TBA) assays. Fluorescence-activated cell sorting (FACS) is an
efficient way of categorizing and quantifying cells based on the status as healthy,
dead, apoptotic, or necrotic. It can distinguish alterations in the cell cycle by
measuring parameters like hypodiploid DNA which is indirect
™
indicative of
apoptosis.
Other pathway-sensitive cell viability assays are enzyme-linked immunosorbent
assay (ELISA) to quantify fragmented DNA, comet assay to detect DNA fragmen-
tation by gel electrophoresis, Caspase Glo3/7 indication of mitochondrial apoptotic
pathway, Hoechst-DNA, and TUNEL assays (TdT-mediated dUTP-biotin nick end
labeling) measure fragmented DNA. Old to new ELISA assay version allows direct
detection of hundreds of cytokine protein in multiplexed assay formats including
8 (LINCOplex). Change patterns in gene/protein expression can be detected by
polymerase chain reaction (PCR), or real-time PCR (qPCR) for quantitative deter-
mination, western blotting, and total protein assay (i.e. BCA or Bradford). This type
of assay can give indication on the modulation of CDKN1A (cell-cycle arrest gene),
GADD45
β
(DNA damage-dependent), IL-6 and NF
κ
BIA (inflammatory response),
