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Traditional and emerging methods for analyzing cell activity in cell culture

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Methods in Cell Science

Abstract

The selection of appropriate techniques to assay for markers of cell activity is important for obtaining optimal results in cell culture-based research. This paper is intended as a guide to many of the assays currently available and new techniques that have been recently introduced in the literature. This paper addresses both manual assay techniques, including the use of hemocytometers, phase contrast microscopy, cell staining, and the immunofluorescent antibody assay (IFA), and automated assays for cell activity, including stained optical density, proliferating cell nuclear antigen, creatine kinase assay, DNA quantification, electronic cell counting, flow cytometry, magnetic cell sorting, image analysis, chemiluminescence, radioisotope labeling, precursor incorporation, in-situ hybridization/ligand binding, and enzyme-linked immuno-culture assay (ELICA). Advantages/disadvantages and applicability of these assays to different areas of cell culture research are discussed, and guidelines for selecting an appropriate assay are suggested.

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References

  1. Aikens RS, Agard DA, Sedat JW (1989). Solid-state imagers for microscopy. Methods Cell Biol 29: 291-313.

    Google Scholar 

  2. Allen RE, Merkel RA, Young RB (1979). Cellular aspects of muscle growth: Myogenic cell proliferation. J Anim Sci 49: 115-127.

    Google Scholar 

  3. Allen RE, Rankin LL, Greene EA, Boxhorn LK, Johnson SE, Taylor RG, Pierce PR (1991). Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells. J Cellular Physiol 165: 307-312.

    Google Scholar 

  4. Allen RK, Reddick TT, Popp DM, Donnell R, Freeman MB, Stevens SL, Goldman MH (1998). Fluorescent activated cell sorting to isolate canine microvascular endthelial cells from adipose tissue. Meth Cell Sci 19: 285-294.

    Google Scholar 

  5. Allen RE, Sheehan SM, Taylor RG, Kendall TL, Rice GM (1995). Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro. J Cellular Phyiol 165: 307-312.

    Google Scholar 

  6. Avrameas S (1992). Amplification systems in immunoenzymatic techniques. J Immunological Meth 150: 23-32.

    Google Scholar 

  7. Bader D, Masaki T, Fischman DA (1982). Immunochemical analysis of myosin heavy chain during avian mygenesis in vivo or in vitro. J Cell Biol 95: 763-770.

    Google Scholar 

  8. Barak LS, Yocum RR, Nothnagel EA, Webb WW (1980). Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazolephallacidin. Proc Natl Acad Sci USA 77(2): 980-984.

    Google Scholar 

  9. Baserga R, Malamud D (1969). Autoradiography: Techniques and application. Modern Methods in Experimental Pathology, p 24. New York: Hoeber Medical Division, Harper and Row.

    Google Scholar 

  10. Bischoff R, Holtzer H (1970). Inhibition of myoblast fusion after one round of DNA synthesis in 5-bromodeoxyuridine. J Cell Biol 44: 134-150.

    Google Scholar 

  11. Blank RS, Thompson MM, Owens GK (1988). Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle. J Cell Biol 107(1): 299-306.

    Google Scholar 

  12. Blanton JR Jr, Grant AL, McFarland DC, Robinson JP, Bidwell CA (1999). Isolation of two populations of myoblasts from porcine skeletal muscle. Muscle Nerve 22(1): 43-50.

    Google Scholar 

  13. Boyd MR (1989). Status of the NCI preclinical antitumor drug discovery screen. Prin Prac Oncol 103: 421-431.

    Google Scholar 

  14. Bronstein I, Edwards B, Voyta JC (1989). 1,2-dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays. J Biolumin Chemilumin 4(1): 99-111.

    Google Scholar 

  15. Byrne KM, Cheng X, Vierck J, Ericson S, Greene EA, Duckett S, Dodson MV (1998). Use of a 96-well plate reader to evaluate proliferation of equine satellite cell clones in vitro. Meth Cell Sci 19(4): 311-316.

    Google Scholar 

  16. Byrne KM, Davis WC, Holmes MA, Brassfield AL, McGuire TC (1997). Cytokine RNA expression in an equine CD4+ subset differentiated by expression of a novel 46-kDa surface protein. Vet Immunol Immunopathol 56: 191-204.

    Google Scholar 

  17. Chen C, Li J, Micko CJ, Pierce GF, Cunningham MR, Lumsden AB (1998). Cytotoxic effects of basic FGF and heparin binding EGF conjugated with cytotoxin saporin on vascular cell cultures. J Surg Res 75(1): 35-41.

    Google Scholar 

  18. Clements JD and Buzy JM (1991). Automated image analysis for counting unstained cultured neurons. J Neurosci Meth 36(1): 1-8.

    Google Scholar 

  19. Collins CL, Wasa M, Souba WW, Abcouwer SF (1998). Determinants of glutamine dependence and utilization by normal and tumor-derived breast cell lines. J Cell Physiol 176(1): 166-178.

    Google Scholar 

  20. Cornelison DW, Wold BJ (1997). Single cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191: 270-283.

    Google Scholar 

  21. Dignass AU, Stow JL, Babyatsky MW (1996). Acute epithelial injury in the rat small intestine in vivo is associated with expanded expression of transforming growth factor alpha and beta. Gut 38(5): 687-693.

    Google Scholar 

  22. Dodson MV, McFarland DC, Bandman E, Dayton W, Yablonka-Reuveni Z, Greene E, Doumit ME, Bergen W, Merkel R, Vierck J, Velleman SG, Koumans J (1995). Status of satellite cell research in agriculture. Basic Appl Myol 5: 5-10.

    Google Scholar 

  23. Dodson MV, McFarland DC, Grant AL, Doumit ME, Velleman SG (1996). Extrinsic regulation of domestic animal-derived satellite cells. Domestic Animal Endocrinology 13(2): 107-126.

    Google Scholar 

  24. Donahue RE, Byrne ER, Thomas TE, Kirby MR, Agricola BA, Sellers SE, Gaudernack G, Karisson S, Lansdorp PM (1996). Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells. Blood 88(11): 4166-4172.

    Google Scholar 

  25. el-Salhy M, Sandström O, Näsström E, Mustajbasic M, Zachrisson S (1997). Application of computer image analysis in endocrine cell quantification. Histochem J 29(3): 249-256.

    Google Scholar 

  26. Florini JR (1989). Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nm. Anal Biochem 182: 399-404.

    Google Scholar 

  27. Frank AJ, Proctor SJ, Tilby MJ (1996). Detection and quantification of melphalan-DNA adducts at the single cell level in hematopoietic tumor cells. Blood 88(3): 977-984.

    Google Scholar 

  28. George-Weinstein M, Gerhart J, Foti GJ, Lash JW (1994). Maturation of myogenic and chondrogenic cells in the presomitic mesoderm of the chick embryo. Exp Cell Res 211: 263-274.

    Google Scholar 

  29. George-Weinstein M, Gerhart J, Reed R, Flynn J, Callihan B, Mattiacci M, Miehle C, Foti GJ, Lash JW, Weintraub H (1996). Skeletal Myogenesis: The preferred pathaway of chick embryo epiblasts cells in vitro. Dev Biol 173: 279-291.

    Google Scholar 

  30. Gillespie PG, Hudspeth AJ (1991). Chemiluminescence detection of proteins from single cells. Proc Nat Acad Sci 88: 2563-2567.

    Google Scholar 

  31. Harlow E, Lane D (1988). Antibodies: A laboratory manual, p 409. New York: Cold Spring Harbor Laboratory.

    Google Scholar 

  32. Helmrich A, Barnes D (1998). Animal cell culture equipment and techniques. Methods Cell Biol 57: 3-17.

    Google Scholar 

  33. Hertzenberg LA, Sweet RG, Hertzenberg LA (1976). Fluorescence-activated cell sorting. Sci Am 234: 108-117.

    Google Scholar 

  34. Hinegardner RT (1971). An improved fluorometric assay for DNA. Analyt Biochem 39: 197-201.

    Google Scholar 

  35. Hofland LJ, De Herder WW, Visser-Wisselaar HA, Van Uffelen C, Waaijers M, Zuyderwijk J, Uitterlinden P, Kros MJ, Van Koetsveld PM, Lamberts SW (1997). Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary-and islet cell tumors. J Clin Endocrinol Metab 82(9): 3011-3018.

    Google Scholar 

  36. Ide M, Jimbo M, Yamamoto M, Kubo O (1997). Tumor cell counting using an image analysis program for MIB-1 immunohistochemistry. Neurol Med Chir 37(2): 158-162.

    Google Scholar 

  37. Ishaque A, Al-Rubeai M (1998). Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J Immunol Methods 221(1-2): 43-57.

    Google Scholar 

  38. Johnson-Wint B, Hollis S (1982). A rapid in situ deoxyribonucleic acid assay to determine cell number in culture and tissue. Anal Biochem 122: 338-344.

    Google Scholar 

  39. Kato K, Hamaguchi Y, Fukui H, Ishikawa E (1975). Enzyme-linked immunoassay. II. A simple method for synthesis of the rabbit antibody-β-galactosidase complex and its general applicability. J Biochem 78: 423-431.

    Google Scholar 

  40. Life Technologies Reference Guide (1999). Compounds for Immunodetection. New York: Life Technologies, R-17, R-18.

    Google Scholar 

  41. Lindon C, Montarras D, Pinset C (1998). Cell cycleregulated expression of the muscle determination factor Myf5 in proliferating myoblasts. J Cell Biol 140(1): 111-118.

    Google Scholar 

  42. Lindsay CD, Hambrook JL, Lailey AF (1997). Monoisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard. Hum Exp Toxicol 16(11): 636-644.

    Google Scholar 

  43. Magun R, Boone DL, Tsang BK, Sorisky A (1998). The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation. Int J Obes Relat Metab Disord 22(6): 567-571.

    Google Scholar 

  44. Matejka GL, Thornemo M, Kernholt A, Lindahl A (1998). Expression of Id-1 mRNA and protein in the post-ischemic regenerating rat kidney. Exp Nephrol 6(3): 253-264.

    Google Scholar 

  45. Marquez LA and Dunford HB (1997). Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient-and steadystate kinetics. Biochemistry 36(31): 9349-9355.

    Google Scholar 

  46. Matsuda R and Abe M (1997). Unfused C2C12 mouse skeletal muscle cells express neurofilament 140k protein. Cell Structure and Function 22: 117-121.

    Google Scholar 

  47. McKay BS, Burke JM (1994). Separation of phenotypically distinct subpopulations of cultured human retinal pigment epithelial cells. Exp Cell Res 213(1): 85-92.

    Google Scholar 

  48. McKeehan WL, McKeehan KA, Hammond SL, Ham RG (1977). Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13(7): 399-416.

    Google Scholar 

  49. Mettenleiter TC, Grawe W (1996). Video imaging of firefly luciferase activity to identify and monitor herpesvirus infection in cell culture. J Virol Meth 59(1-2): 155-160.

    Google Scholar 

  50. Nixon AJ, Lillich JT, Burton-Wurster N, Lust G, Mohammed HO (1998). Differentiated cellular function in fetal chondrocytes cultured with insulinlike growth factor-I and transforming growth factorbeta. J Orthop Res 16(5): 531-541.

    Google Scholar 

  51. Ogata K, Kurki P, Celis JE, Nakamura RM, Tan EM (1987). Monoclonal antibodies to a nuclear protein (PCNA/cyclin) associated with DNA replication. Exp Cell Res 168(2): 475-486.

    Google Scholar 

  52. Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent GJ (1989). A rapid and convenient assay for counting cells cultured in microwell plates: Application for assessment of growth factors. J Cell Sci 92: 513-518.

    Google Scholar 

  53. Oshiro Y, Gukuda T, Tsuneyoshi M (1994). Fibrosarcoma versus fibromatoses and cellular nodular fasciitis. A comparative study of their proliferative activiey using proliferating cell nuclear antigen, DNA flow cytometry, and p53. Am J Surg Pathol 18(7): 712-719.

    Google Scholar 

  54. Padubidri A and Browne E Jr (1996). Effect of vascular endothelial growth factor (VEGF) on survival of random extension of axial pattern skin flaps in the rat. Ann Plast Surg 37(6): 604-611.

    Google Scholar 

  55. Pizzonia JH, Gesek FA, Kennedy SM, Coutermarach BA, Bacskal BJ, Friedman PA (1991). Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev Biol 27A: 409-416.

    Google Scholar 

  56. Plumb JA, Milroy R, Kaye SB (1989). Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazoliumbased assay. Cancer Res 49(16): 4435-4440.

    Google Scholar 

  57. Porstmann T, Kiessig ST (1992). Enzyme immunoassay techniques: An overview. J Immunological Methods 150: 5-21.

    Google Scholar 

  58. Reynolds JE, Li H, Eastman A (1996). Detection of apoptosis by flow cytometry of cells simultaneously stained for intracellular pH (carboxy SNARF-1) and membrane permeability (Hoechst 33342). Cytometry 25(4): 349-357.

    Google Scholar 

  59. Sarvazyan N (1998). A new approach to assess viability of adult cardiomyocytes: Computer-assisted image analysis. J Mol Cell Cardiol 30(2): 297-301.

    Google Scholar 

  60. Schaap AP, Akhavan H, Romano LJ (1989). Chemiluminescent substrates for alkaline phosphatase: Application to ultrasensitive enzyme-linked immunoassays and DNA probes. Clin Chem 35(9): 1863-1864.

    Google Scholar 

  61. Scott-Burden T, Tock CL, Bosely JP, Clubb FJ Jr, Parnis SM, Schwarz JJ, Engler DA, Frazier OH, Casscells SW 3rd (1998). Nonthrombogenic, adhesive cellular lining for left ventricular assist devices. Circulation 98(19 Suppl): II339-345.

    Google Scholar 

  62. Shalev A, Greenberg AH, McAlpine PJ (1980). Detection of attograms of antigen by a high-sensitivity enzyme-linked immunoadsorbent assay (Hs-ELISA) using a fluorogenic substrate. J Immunol Methods 38: 125-132.

    Google Scholar 

  63. Singh SP, Jayanth VR Chandna S, Dwarakanath BS, Singh S, Adhikari HS, Jain V (1998). Radioprotective effects of DNA ligands Hoechst-33342 and 33258 in whole body irradiated mice. Indian J Exp Biol 36(4): 375-384.

    Google Scholar 

  64. Skehan P, Storeng R, Scudiero N, et al. (1989). Evaluation of colorimetric and biomass stains for assaying in vitro drug effects upon human tumor cell lines. Proc Am Soc Cancer Res 40: 3259-3267.

    Google Scholar 

  65. Smith CK, Janney MJ, Allen RE (1994). Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells. J Cell Physiol 159: 379-385.

    Google Scholar 

  66. Stewart NT, Byrne K, Ragle C, Dodson MV (in progress). Use of enzyme-linked immuno-culture assay (ELICA) and confocal immunofluorescent microscopy to determine levels of muscle specific markers of differentiation in satellite cell cultures.

  67. Swales NJ, Luong C, Caldwell J (1996). Cryopreservation of rat and mouse hepatocytes. I. Comparative viability studies. Drug Metab Dispos 24(11): 1218-1223.

    Google Scholar 

  68. Tajima S, Tsuji K, Ebihara Y, Sui X, Tanaka R, Muraoka K, Yoshida M, Yamada K, Yasukawa K, Taga T, Kishimoto T, Nakahata T (1996). Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 184(4): 1357-1364.

    Google Scholar 

  69. Tarnowski BI, Sens DA, Nicholson JH, Hazen-Martin DJ, Garvin AJ, Sens MA (1993). Automatic quantitation of cell growth and determination of mitotic index using DAPI nuclear staining. Pediatr Pathol 13(2): 249-265.

    Google Scholar 

  70. Tatsumi R, Anderson JE, Nevoret CJ, Halevy O, Allen RE (1998). HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells. Dev Biol 194: 114-128.

    Google Scholar 

  71. Teare JM, Islam R, Flanagan R, Gallagher S, Davies MG, Grabau C (1997). Measurement of nucleic acid concentrations using the DyNA Quant and the Gene Quant. Biotechniques 22(6): 1170-1174.

    Google Scholar 

  72. Th'eon AP, Metzger L, Griffey S (1994). In situ analysis of cellular proliferation in canine, feline and equine tumors by immunohistochemistry: A comparison of bromodeoxyuridine, proliferating cell nuclear antigen, and interchromatin-associated antigen immunostaining techniques. J Vet Diagn Invest 6(4): 453-457.

    Google Scholar 

  73. Trickett AE, Ford DJ, Lam-Po Tang PRL, Vowels MR (1990). Comparison of magnetic particles for immunomagnetic bone marrow purging using an acute lymphoblastic leukaemia model. Transpl Proc 22: 2177-2178.

    Google Scholar 

  74. Van der Ven PFM, Fürst DO (1997). Assembly of titin, myomesin and m-protein into the sarcmeric M band in differentiating human skeletal muscle cells in vitro. Cell Structure and Function 22: 163-171.

    Google Scholar 

  75. Vierck JL, McNamara JP, Dodson MV (1996). Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle. Methods Cell Sci 18: 309-314.

    Google Scholar 

  76. Watson JV, Erba E (1992). Flow cytometry. In: Freshney RI (ed), Animal cell culture, a practical approach, pp 165-212. Oxford, IRL press at Oxford University Press.

    Google Scholar 

  77. Wheatley DP, Wang YL (1998). Indirect immunofluorescence microscopy in cultured cells. Methods Cell Biol 57: 313-332.

    Google Scholar 

  78. Whitehead TP, Thorpe GHG, Gartes TJN, Groucutt C, Kticka LJ (1983). Enhanced luminescence procedure for sensitive detection of peroxidase-labeled conjugates in immunoassay. Nature 305: 158-166.

    Google Scholar 

  79. Wolf HK, Dittrich KL (1992). Detection of proliferation cell nuclear antigen in diagnostic histopathology. J Histochm Cytochem 40(9): 1269-1273.

    Google Scholar 

  80. Woodruff TK (1998). Cellular localization of mRNA and protein: in situ hybridization histochemistry and in situ ligand binding. Methods Cell Biol 57: 333-351.

    Google Scholar 

  81. Yablonka-Reuveni Z, Nameroff M (1987). Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation. Histochemistry 87(1): 27-38.

    Google Scholar 

  82. Yamada K, Donner DB (1985). Evidence that noncovalent forces, thiol and disulphide groups affect the structure and binding properties of the prolactin receptor on hepatocytes from pregnant rats. Biochem J 228(2): 383-390.

    Google Scholar 

  83. Young RB, Allen RE (1979). Transitions in gene activity during development of muscle fibers. J Animal Sci 48(4): 837-852.

    Google Scholar 

  84. Young HE, Sippel J, Putnam LS, Lucas PA, Morrison DC (1992). Enzyme-linked immuno-culture assay. J Tiss Cult Meth 14: 31-36.

    Google Scholar 

  85. Yun Y, Mcfarland DC, Pesail JE, Gilkerson KK, Vander Wal LS, Ferrin NH (1997). Variation in response to growth factor stimuli in satellite cell populations. Comp Biochem Physiol 117A(4): 463-470.

    Google Scholar 

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Stewart, N.T., Byrne, K.M., Hosick, H.L. et al. Traditional and emerging methods for analyzing cell activity in cell culture. Methods Cell Sci 22, 67–78 (2000). https://doi.org/10.1023/A:1009839501174

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