[求救] 關於epithelial voltmeter
在讀一篇論文
裡面有用到epithelial voltmeter(EVOM)的測量
以及TER值的解讀
可是我實在看不太懂,又不知道上哪找這方面的資料
不知道有沒有高手可以幫幫忙,簡單介紹一下EVOM的原理,
以及TER值所代表的意義?
好像可以用來表示測量細胞層的完整度,是嗎?
論文名稱
a2 but not a1 AMP-activated Protein Kinase Mediates
Oxidative Stress-induced Inhibition of Retinal Pigment
Epithelium Cell Phagocytosis of Photoreceptor Outer Segments
作者 Suofu Qin1 and Gerald W. De Vries
關於實驗結果的片段,節錄如下
No Role of AMPK in Oxidative Stress-induced Breakdown of
RPE Monolayer—Integrity of the RPE monolayer was determined
by measuring TER. RPE cells grown in transwells for 3
days were switched to 1% FBS-containing medium, and progression
of TER was monitored daily up to 1 week. TER reading
showed that resistance reached to a plateau in 3 days and that
no further increase was detected up to 1 week culture (data not
shown). The TER values of ARPE19 monolayer averaged 51.8
ohms/cm2. To evaluate the effect of oxidative stress on monolayer
integrity, the TER was measured in ARPE19 cells exposed
to 1 mM hydrogen peroxide as a function of time up to 6 h. Treatment
with hydrogen peroxide caused a significant decrease in TER
versus untreated control at 3 h (Fig.5A). Further exposure reduced TER
to 23% of control. AMPK knockdown by siRNA did not affect the
TER reading, and the decrease in TER by hydrogen peroxide was also
not altered by AMPK knockdown (Fig. 5A), suggesting that AMPK is
not involved in regulating oxidative stress-induced breakdown of RPE
monolayer.
To confirm no role of AMPK in oxidative stress-induced monolayer
breakdown observed by TER measurement, transepithelial flux assays
were performed. Dextran flux from apical to basolateral side was
increased after hydrogen peroxide treatment (Fig. 5B). There was a
70% increase in fluorescence after 6 h of exposure. Similar to the
TER measurement, knockdown of a2 by siRNA did not
alter the flux rate of dextran through the ARPE19 monolayer before and
after hydrogen peroxide treatment, confirming that AMPK plays no role in regulating breakdown
of RPE monolayer by oxidative stress.
關於材料與方法的片段,節錄如下:
Measurement of Barrier Functions—Approximately 1.78 x
100000 cells/cm2 were seeded in individual transwell filters in 0.5
ml of growth medium (0.4M pore size, 12mmdiameter). 1 ml
of medium was added at the basal chamber to level the height of
the liquid for preventing hydrostatic pressure. Transepithelial
electrical resistance (TER) was measured using an epithelial voltmeter
(EVOM; World Precision Instruments, Sarasota, FL) according
to the manufacturer’s instructions. The cells were taken from the
incubator and placed at room temperature for 30 min of equilibration
before the measurements. The TER (in ohms/cm2) of the filter alone was
measured as background and subtracted from the TERs obtained
with the filters and the RPE cells.
Confluent RPE cells grown in transwell filter inserts were switched to
serum-free medium and were treated with 1 mM hydrogen peroxide.
Measurements were made at 3 and 6 h and were repeated at least 3
times for each well, and 3 different wells were used for each treatment.
Paracellular permeability of ARPE19 monolayer was determined
by measuring the apical-to-basolateral movements of FITC-dextran
(70 kDa). After 6 h of exposure to hydrogen peroxide, 10g/ml FITCdextran
was added to the apical chamber. 100 l of medium was
removed from the basal chamber at 60 min after adding the molecules,
and fluorescence intensity was measured with a fluorometer at an
excitation wavelength of 495 nm and emission wavelength of 525 nm.
感謝!!!
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