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A rapid sampling method for measuring uptake of small molecules by cultured cell monolayers
ANALYTICAL
A Rapid
BIOCHEMISTRY
117, 67-69
Sampling
Method
(1981)
for Measuring
Cultured ROBERT Marrs
McLean
Department
S.
Uptake
of Small
Molecules
by
Cell Monolayers’
SLAUGHTER
AND
of Biochemistry,
EUGENE
Baylor
College
May
7, 1981
Received
M.
BARNES,
of Medicine,
JR.’ Houston,
Texas
77030
A new technique is described which allows determination of solute uptake into cultured cell monolayers at times approaching 0.5 s. The apparatus consists of a section of a plastic tissue culture multidish which is held in a rotatable clamp and a manifold for simultaneous washing of the multidishes with ice-cold buffer. This technique and apparatus was used to measure the initial rates of nucleoside and base transport by Chinese hamster fibroblasts.
The transport of nucleosides and bases by facilitated diffusion into cultured animal cells results in equilibration with the external solute concentration within 1 to 5 min (1,2). In order to determine initial rates of solute uptake, the assay times employed must be within a few seconds so that the uptake values are all in the linear range. Methods have been described for measuring rapid solute uptake into cell monolayers using specific inhibitors or into cell suspensions (2). A general application of the monolayer technique is complicated by the requirement for large quantities of appropriate inhibitors and by low resolution for uptake times less than 4s. This paper reports a method for rapid measurement of uptake rates for substrates by cell monolayers. Initial rates of substrate entry determined from time points over 0.5 to 4.5 s intervals yielded values with a corelation coefficient greater than 0.95. MATERIALS
AND
dishes (Costar, Cambridge, Mass.) using Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Each well ( 16mm diameter) was inoculated with 6 X lo4 cells and grown at 37°C in a 5% CO2 atmosphere for 3 days or until near confluence. For transport assays the 24-well multidishes were cut with an electrically heated tungsten wire into three rectangular sections (4.2 x 8.5 cm) of 8 wells each. These sections were washed immediately before use with a Hepes3-buffered saline (per liter: 7.1 g NaCl, 0.4 g KCl, 0.2 g CaCL 0.2 g MgS04.7Hz0, 0.125 g NaH2P04.H20, and 6.0 g Hepes adjusted to pH 7.4 with NaOH). This saline wash was equilibrated at 30°C and then delivered with a manifold illustrated in Fig. 1. The manifold was constructed by cementing eight plastic pipet tips (C-200, Rainin Instrument Company, Brighton Mass.) onto a 25-cm* tissue culture T flask (Model 3013, Falcon, Oxnard, Calif.). The pipet tips were cut to give a 2-mm internal diameter and affixed over holes (5-mm diameter, 22 mm center to center) perforated into the flask with a heated metal rod. The array of these nozzles (Fig. 1) on the manifold matched the centers of the eight wells of the multidish section.
METHODS
Chinese hamster lung fibroblasts were cultured on Cluster-24 tissue culture multi’ This work was supported by Grant AM 17436 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. * Supported by Research Career Development Award AM 00052 from the National Institute of Arthritis, Metabolism, and Digestive Diseases.
’ Abbreviation used: perazineethanesulfonic 67
Hepes, acid.
0003-2697/S
4-(2-hydroxyethyl)-l-piI /150067-03%02.00/O
Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
68
SLAUGHTER
w&h
AND
BARNES
manifold
FIG. 1. Apparatus for measurement of uptake of small molecules washed eight-well section cut from a tissue culture multidish was held frame. The assay was initiated by addition of assay mixture containing The uptake of substrate was terminated by pivoting the multidish delivered an ice-cold stop solution (Materials and Methods).
The washed multidish section was then placed in a wire clamp which held it in a 30°C water bath. The uptake assay was initiated by sequential addition at 0.5 or l.Os intervals of 0.4 ml assay mixture containing 4.4 &i of [3H]hypoxanthine or [methyf3H]thymidine in Hepes-buffered saline. Each assay mixture was equilibrated at 30°C and
by cultured cell monolayers. A in a 30°C water bath by a wire radioactively labeled substrates, over a washing manifold which.
rapidly added to the wells by means of a repeating syringe dispenser (Tridak, Brookfield Center, Conn.). At the end of the assay period, usually 4.5 to 9 s, the handle was rotated thus flipping the holder and attached multidish over the wash manifold (Fig. 1) which delivered an ice-cold stop solution (per liter: 8.6 g NaCl, 0.1 g MgC12.6H20, 0.158
2I B” E 50 5 5c .40 I= 2 .30 ti g E
.20
d t
.lO
1
2
3
4
2
4
6
6
TIME bed
FIG. 2. Transport of thymidine and hypoxanthine by Chinese hamster lung fibroblasts (RJK688). The assay was carried out as described in the legend to Fig. I. The final concentrations of [‘Hlthymidine (left panel) were (from bottom to top) 100, 200, 300, and 900 fic~ and for hypoxanthine (right panel, bottom to top) 100, 200, 400, and 800 FM.
DETERMINATION
OF
SOLUTE
UPTAKE
g NaH2P04.H20, 0.938 g Na2HP04, 0.2 g KCl, 0.1 g CaC&). A total volume of 80- 100 ml of stop solution was delivered during a 2- to 3-s wash procedure. In order to prevent escape of radioactive material, the apparatus was screened with a plastic sheet and placed in a large plastic tray. After blotting the multidish section on paper towels, 1 ml of 0.2 M NaOH was added to each well and the section was incubated for 40 min at 65°C. A 600-~1 aliquot of this digest was transferred to a counting vial and neutralized with 400 ~1 of 2.5% acetic acid. Samples were counted by liquid scintillation in ACS counting solution (Amersham, Arlington Heights, Ill.). A IOO~1 aliquot was taken for protein determination (3). All values for solute uptake were expressed as nanomoles per milligram of cell protein.
INTO
CULTURED
CELL
MONOLAYERS
69
say mixture or nonspecific binding. Neither the slopes or intercepts were altered significantly if the volume of wash solution or time of washing was doubled. The kinetics of nucleoside and base transport have been studied with this method (1). The Chinese hamster lung fibroblast strain (RJK688) employed in these and the present studies was deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Use of this strain insures that transport rather than metabolism is rate-limiting in the uptake measurements. However, with the improvements in time resolution obtained as reported here, it should be possible to estimate transport rates for these substrates into wild-type cells. Indeed, this method seems broadly applicable to measurement of rates for rapid binding or translocation of radioactively labeled substrates by monolayers of cultured cells.
RESULTS AND DISCUSSION
The technique of sequential additions of assay mixture to an eight-well multidish allows determination of uptake rates of hypoxanthine and thymidine by fibroblasts as shown in Fig. 2. The experimental values for solute entry are proportional with time for at least 4.5 or 9 s, respectively, for thymidine or hypoxanthine uptake. The slopes are, then, the initial velocities. The intercepts increase with increasing substrate concentration as would be expected from residual as-
REFERENCES 1. Slaughter, R. S., Fenwick, R. G., Jr., and Barnes, Jr., E. M., (1981)Arch. Biochem. Biophys. 211, 494-499. 2. Plagemann, P. G. W., and Wohlhueter, R. M. (1980) in Current Topics in Membranes and Transport (Bronner, F., and Kleinzeller, A., eds.), Vol. 14, pp. 225-330, Academic Press, New York. 3. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265219.
A Rapid
BIOCHEMISTRY
117, 67-69
Sampling
Method
(1981)
for Measuring
Cultured ROBERT Marrs
McLean
Department
S.
Uptake
of Small
Molecules
by
Cell Monolayers’
SLAUGHTER
AND
of Biochemistry,
EUGENE
Baylor
College
May
7, 1981
Received
M.
BARNES,
of Medicine,
JR.’ Houston,
Texas
77030
A new technique is described which allows determination of solute uptake into cultured cell monolayers at times approaching 0.5 s. The apparatus consists of a section of a plastic tissue culture multidish which is held in a rotatable clamp and a manifold for simultaneous washing of the multidishes with ice-cold buffer. This technique and apparatus was used to measure the initial rates of nucleoside and base transport by Chinese hamster fibroblasts.
The transport of nucleosides and bases by facilitated diffusion into cultured animal cells results in equilibration with the external solute concentration within 1 to 5 min (1,2). In order to determine initial rates of solute uptake, the assay times employed must be within a few seconds so that the uptake values are all in the linear range. Methods have been described for measuring rapid solute uptake into cell monolayers using specific inhibitors or into cell suspensions (2). A general application of the monolayer technique is complicated by the requirement for large quantities of appropriate inhibitors and by low resolution for uptake times less than 4s. This paper reports a method for rapid measurement of uptake rates for substrates by cell monolayers. Initial rates of substrate entry determined from time points over 0.5 to 4.5 s intervals yielded values with a corelation coefficient greater than 0.95. MATERIALS
AND
dishes (Costar, Cambridge, Mass.) using Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Each well ( 16mm diameter) was inoculated with 6 X lo4 cells and grown at 37°C in a 5% CO2 atmosphere for 3 days or until near confluence. For transport assays the 24-well multidishes were cut with an electrically heated tungsten wire into three rectangular sections (4.2 x 8.5 cm) of 8 wells each. These sections were washed immediately before use with a Hepes3-buffered saline (per liter: 7.1 g NaCl, 0.4 g KCl, 0.2 g CaCL 0.2 g MgS04.7Hz0, 0.125 g NaH2P04.H20, and 6.0 g Hepes adjusted to pH 7.4 with NaOH). This saline wash was equilibrated at 30°C and then delivered with a manifold illustrated in Fig. 1. The manifold was constructed by cementing eight plastic pipet tips (C-200, Rainin Instrument Company, Brighton Mass.) onto a 25-cm* tissue culture T flask (Model 3013, Falcon, Oxnard, Calif.). The pipet tips were cut to give a 2-mm internal diameter and affixed over holes (5-mm diameter, 22 mm center to center) perforated into the flask with a heated metal rod. The array of these nozzles (Fig. 1) on the manifold matched the centers of the eight wells of the multidish section.
METHODS
Chinese hamster lung fibroblasts were cultured on Cluster-24 tissue culture multi’ This work was supported by Grant AM 17436 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. * Supported by Research Career Development Award AM 00052 from the National Institute of Arthritis, Metabolism, and Digestive Diseases.
’ Abbreviation used: perazineethanesulfonic 67
Hepes, acid.
0003-2697/S
4-(2-hydroxyethyl)-l-piI /150067-03%02.00/O
Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
68
SLAUGHTER
w&h
AND
BARNES
manifold
FIG. 1. Apparatus for measurement of uptake of small molecules washed eight-well section cut from a tissue culture multidish was held frame. The assay was initiated by addition of assay mixture containing The uptake of substrate was terminated by pivoting the multidish delivered an ice-cold stop solution (Materials and Methods).
The washed multidish section was then placed in a wire clamp which held it in a 30°C water bath. The uptake assay was initiated by sequential addition at 0.5 or l.Os intervals of 0.4 ml assay mixture containing 4.4 &i of [3H]hypoxanthine or [methyf3H]thymidine in Hepes-buffered saline. Each assay mixture was equilibrated at 30°C and
by cultured cell monolayers. A in a 30°C water bath by a wire radioactively labeled substrates, over a washing manifold which.
rapidly added to the wells by means of a repeating syringe dispenser (Tridak, Brookfield Center, Conn.). At the end of the assay period, usually 4.5 to 9 s, the handle was rotated thus flipping the holder and attached multidish over the wash manifold (Fig. 1) which delivered an ice-cold stop solution (per liter: 8.6 g NaCl, 0.1 g MgC12.6H20, 0.158
2I B” E 50 5 5c .40 I= 2 .30 ti g E
.20
d t
.lO
1
2
3
4
2
4
6
6
TIME bed
FIG. 2. Transport of thymidine and hypoxanthine by Chinese hamster lung fibroblasts (RJK688). The assay was carried out as described in the legend to Fig. I. The final concentrations of [‘Hlthymidine (left panel) were (from bottom to top) 100, 200, 300, and 900 fic~ and for hypoxanthine (right panel, bottom to top) 100, 200, 400, and 800 FM.
DETERMINATION
OF
SOLUTE
UPTAKE
g NaH2P04.H20, 0.938 g Na2HP04, 0.2 g KCl, 0.1 g CaC&). A total volume of 80- 100 ml of stop solution was delivered during a 2- to 3-s wash procedure. In order to prevent escape of radioactive material, the apparatus was screened with a plastic sheet and placed in a large plastic tray. After blotting the multidish section on paper towels, 1 ml of 0.2 M NaOH was added to each well and the section was incubated for 40 min at 65°C. A 600-~1 aliquot of this digest was transferred to a counting vial and neutralized with 400 ~1 of 2.5% acetic acid. Samples were counted by liquid scintillation in ACS counting solution (Amersham, Arlington Heights, Ill.). A IOO~1 aliquot was taken for protein determination (3). All values for solute uptake were expressed as nanomoles per milligram of cell protein.
INTO
CULTURED
CELL
MONOLAYERS
69
say mixture or nonspecific binding. Neither the slopes or intercepts were altered significantly if the volume of wash solution or time of washing was doubled. The kinetics of nucleoside and base transport have been studied with this method (1). The Chinese hamster lung fibroblast strain (RJK688) employed in these and the present studies was deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Use of this strain insures that transport rather than metabolism is rate-limiting in the uptake measurements. However, with the improvements in time resolution obtained as reported here, it should be possible to estimate transport rates for these substrates into wild-type cells. Indeed, this method seems broadly applicable to measurement of rates for rapid binding or translocation of radioactively labeled substrates by monolayers of cultured cells.
RESULTS AND DISCUSSION
The technique of sequential additions of assay mixture to an eight-well multidish allows determination of uptake rates of hypoxanthine and thymidine by fibroblasts as shown in Fig. 2. The experimental values for solute entry are proportional with time for at least 4.5 or 9 s, respectively, for thymidine or hypoxanthine uptake. The slopes are, then, the initial velocities. The intercepts increase with increasing substrate concentration as would be expected from residual as-
REFERENCES 1. Slaughter, R. S., Fenwick, R. G., Jr., and Barnes, Jr., E. M., (1981)Arch. Biochem. Biophys. 211, 494-499. 2. Plagemann, P. G. W., and Wohlhueter, R. M. (1980) in Current Topics in Membranes and Transport (Bronner, F., and Kleinzeller, A., eds.), Vol. 14, pp. 225-330, Academic Press, New York. 3. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265219.