Summary
Methods have been developed for measuring the intracellular water space (ICS) in cultured diploid fibroblast monolayers. The values obtained have been compared to similar measurements of ICS in suspended fibroblasts using an oil spin method. Markers commonly used for measurement of total water space (TWS) ([3H]H2O, [14C]urea) and extracellular space (ECS) ([3H]inulin, [14C]l-glucose, [3H]sucrose, [3H]d-mannitol) were investigated for use in cell monolayers. Monolayer incubations were terminated by rapidly rinsing the culture dishes three times with ice cold buffer. The distribution volume of the TWS marker [14C]urea plateaued at 10 to 15 min and was independent of urea concentration. [3H]H2O was not a suitable marker for measuring total water space in cell monolayers because of rapid loss of intracellular label during rinsing. Intracellular space was calculated by subtracting [3H]sucrose space (5 min) from [14C]urea space (20 min) after incubation of fibroblasts with both markers. Values obtained for ICS (mean ± SE;µl/106 cells) of fibroblasts from two cell lines measured in monolayer (1.74±0.11 and 1.60±0.10) and in suspension (1.88±0.07 and 1.78±0.11) was approximately 10% smaller than the values for cell size (2.01 and 2.22) obtained from Coulter Counter sizing. Thus, the methods developed for measurement of ICS in monolayer fibroblasts yield data comparable to those obtained with the more standard oil spin method. Furthermore, the methods can be applied to measurement of ICS in other types of adherent cells.
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Howard, B. V.; Mott, D. M.; Fields, R. M.; Bennett, P. H. Insulin stimulation of glucose entry in cultured human fibroblasts. J. Cell Physiol. 101: 129–138; 1979.
Hidaka, H.; Howard, B. V.; Kosmakos, F. C.; Fields, R. M.; Craig, J. W.; Bennett, P. H.; Larner, J. Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts. Diabetes 29: 806–810; 1980.
Ishibashi, F.; Hidaka, H.; Bennett, P. H.; Howard, B. V. Glucose enhancement of insulin action—elevated glucose levels increase insulin stimulation on 2-deoxyglucose uptake in cultured human fibroblasts. J. Clin. Endocrinol. Metab. 54: 34–39; 1981.
Fujimoto, W. Y.; Williams, R. H. Insulin action on cultured human fibroblasts. Glucose uptake, protein synthesis, RNA synthesis. Diabetes 23: 443–448; 1974.
Germinario, R. J.; Oliveria, M. Stimulation of hexose transport in cultured human skin fibroblasts by insulin. J. Cell. Physiol. 99: 313–318; 1979.
Podskalny, J. M.; Kahn, C. R. Insulin activates glycogen synthase in cultured human fibroblasts. Diabetes 29: 724–729; 1980.
Berhans, P.; Olefsky, J. M. Effects of insulin and insulin-like agents on the glucose transport system of cultured human fibroblasts. Diabetes 30: 523–529; 1981.
Gliemann, J.; Osterlind, K.; Vinten, J.; Gammeltofts, S. A procedure for measurement of distribution spaces in isolated fat cells. Biochim. Biophys. Acta 286: 1–9; 1972.
Thorsteinsson, D.; Gliemann, J.; Vinten, J. The content of water and potassium in fat cells. Biochim. Biophys. Acta 428: 223–227; 1976.
McDaniel, M. L.; Anderson, S.; Fink, J.; Roth, C.; Lacy, P. E. Effect of alloxan on permeability and hexose transport in rat pancreatic islets. Endocrinology 97: 68–75; 1975.
Andreasen, P. A.; Schaumburg, B. P.; Osterlind, K.; Vinten, J.; Gammeltoft, S.; Gliemann, J. A rapid technique for separation of thymocytes from suspensions by centrifugation through oil. Anal. Biochem. 59: 610–616; 1974.
Werdan, K.; Lehner, K.; Cremer, T.; Stevenson, A. F. G.; Messerschmidt, O.d-Glucose transport into suspended human fibroblasts. Hoppe Seylers Z. Physiol. Chem. 361: 91–104; 1980.
Howard, B. V.; De La Llera, M.; Howard, W. J. A new method for the establishment of diploid fibroblast cell cultures from human foreskins. Proc. Soc. Exp. Biol. Med. 153: 280–283; 1976.
Howard, B. V.; Fields, R. M.; Mott, D. M.; Savage, P. J.; Nagulesparan, M.; Bennett, P. H. Diabetes and cell growth—lack of differences in growth characteristics of fibroblasts from diabetic and non-diabetic Pima Indians. Diabetes 29: 119–124; 1980.
Houghton, B. A.; Stidworthy, G. H. A growth history comparison of human diploid cells WI-38 and IMR-90: proliferative capacity and cell sizing analysis. In Vitro 15: 697–702; 1979.
Cristofalo, V. J.; Kritchevsky, D. Cell size and nucleic acid content in diploid human cell line WI-38 during aging. Medicina Experimentalis 19 (6): 313–320; 1969.
Bowman, P. D.; Daniel, C. W. Characteristics of proliferative cells from young, old and transformed WI-38 cultures. Adv. Exp. Med. Biol. 53: 107–122; 1975.
Cremer, T.; Werdan, K.; Stevenson, A. F. G.; Lehner, K.; Messerschmidt, O. Aging in vitro andd-glucose uptake kinetics of diploid human fibroblasts. J. Cell. Physiol. 106: 99–108; 1981.
Ishibashi, F.; Howard, B. V. Alloxan and H2O2 action on glucose metabolism in cultured fibroblasts. J. Biol. Chem. 256: 12134–12139; 1981.
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This work was supported in part by funds from the National Institutes of Health Contract N01-AM-9212.
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Vasquez, B., Ishibashi, F. & Howard, B.V. Measurement of intracellular volume in monolayers of cultured cells. In Vitro Cell.Dev.Biol. -Plant 18, 643–649 (1982). https://doi.org/10.1007/BF02796397
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DOI: https://doi.org/10.1007/BF02796397