Reference
Lee, J-S, et. al.
Molecular Pharmacology 46:627-638(1994).
Method
Suspensions of logarithmic phase cells were obtained from tissue culture plates by trypsinization. During
the accumulation period, four aliquots of cells were resuspended in rhodamine-containing medium ( Improved
minimum essential medium with 10% fetal calf serum and 0.5 µ g/ml rhodamine 123 ) and incubated with or
without 3.0 µ g/ml cyclosporin A at 37 ° in 5% CO
2 for 30 min. After the accumulation
period, efflux was initiated by sedimentation at 600 ×
g and resuspension in rhodamine-free
medium ( Improved minimum essential medium with 10% fetal calf serum ), with or without 3.0 µ g/ml
cyclosporin A. The efflux was carried out at 37 ° in 5% CO
2 for 90 min. At the end of both the
accumulation and efflux periods, cells were sedimented, wqashed in ice-cold Hank's buffered salt solution, placed
in Hank's buffered salt solution with 10% fetal calf serum on ice, and kept in the dark until flow cytometric
analysis. Samples were analysed on a FACScan flow cytometer ( Becton Dickinson ) equipped with a 488-nm argon
laser. The green fluorescence of rhodamine 123 was collected after a 530-nm band pass filter. Samples were gated
on forward scatter versus side scatter to exclude cell debris and clumps. A minimum of 6000 events were collected
for each sample. Histograms were generated for each cell line and the median rhodamine fluorescence obtained for
each peak were identified by channel number, and this number ( denoting the logarithm of fluorescence intensity )
was used to indicate the amount of rhodamine in the cells in each incubation condition. A difference of 256
channel numbers represents a 10-fold change in fluorescence intensity.
Units
The rhodamine efflux value is taken to be the difference in channel number
in the presence and absence of cyclosporin A.
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Pgp measurement by PCR analysis
Reference
Alvarez, M., et. al.,
Journal of Clinical Investigation,
95:2205-2214(1995).
Method
Quantitative PCR for
mdr-1 and beta-2 microglobulin expression was performed using published
primers as previously described (Murphy, et. al.,
Biochemistry
29:10351-10356(1990). ) Briefly, in an initial experiment, 125 ng of RNA was reverse transcribed
and amplified for 30 cycles to obtain an estimate of
mdr-1 expression. With 125ng of RNA,
mdr-1 expression was detected in 28 of 60 samples. Precise quantitation was then performed for 28
positive cell lines using serially diluted samples. Expression was examined in the 32 negative cell lines by
using 1 µ g of RNA. If PCR was negative at 30 cycles, the number of cycles was increased to 40. RNA
isolated from SW620 human colon carcinoma cells cultured in this laboratory was used as a reference standard.
Expression od
mdr-1 can be readily detected in 125 ng of RNA and in serial dilutions below this. At
these dilutions, PCR is in the exponential range. RNA from the SW620 cell line was serially diluted and amplified
in every experiment, and a sample was included in every gel. Thus, in every experiment, the reaction conditions
were internally controlled, and in every gel a reference standard was included. The level in the SW620 cell line
was arbitrarily assigned a value of 1 and all other values were determined relative to this. This allowed for
quantitation based on either measured RNA or beta-2 microglobulin levels. In the latter, the
mdr-1
values obtained the exponential range of amplification were divided by the beta-2 microglobulin values similarly
obtained; the
mdr-1 level of the SW620 cell line was 10 (
mdr-1/beta-2 = 10/1 = 10).
All quantitations were performed by densitometry.
md1 level relative to SW620
Standardization of results by RNA concentration is linearly related to standardization of results by beta-2
microglobulin expression
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PCR analysis of Pgp Expression
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