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Fumiko Kashiwagi, Kenji Kashiwagi, Yoko Iizuka, Shigeo Tsukahara; Effects of Brain-Derived Neurotrophic Factor and Neurotrophin-4 on Isolated Cultured Retinal Ganglion Cells: Evaluation by Flow Cytometry. Invest. Ophthalmol. Vis. Sci. 2000;41(8):2373-2377.
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purpose. Effects of brain-derived neurotrophic factor (BDNF) and neurotrophin
(NT)-4 on retinal ganglion cells (RGCs) isolated and cultured in a
serum-free medium are evaluated objectively by using flow cytometry.
methods. RGCs from the retinas of 2-day-old rats were isolated in a two-step
panning and cultured in a serum-free medium. BDNF (1, 10, and 100 pg/ml
or 1, 10, and 100 ng/ml), NT-4 (0.1, 1, 10, and 100 ng/ml) or their
vehicle, phosphate-buffered saline, were individually added to aliquots
of the medium to be cultured for 48 hours. Then, after adding
5-chloromethylfluorescein diacetate, the survival of RGCs was evaluated
using flow cytometry.
results. The method used allowed the authors to analyze 10,000 RGCs per
sample in approximately 2 minutes, so that a much larger number of
cells was evaluated in a shorter period than with previously reported
methods. RGCs were classified into either large or small RGCs, and the
survival of each of these groups was determined objectively by the
amount of fluorescent emission. BDNF improved the survival rate of RGCs
concentration-dependently. In particular, the survival rate of small
RGCs was greatly improved. BDNF at 100 ng/ml increased the survival
rate of small RGCs by 17.4% and that of large RGCs by 7.8% in
comparison to the controls. NT-4 did not significantly improve the
survival rates of either large or small RGCs.
conclusions. BDNF improved the survival rate of RGCs, particularly of small RGCs,
concentration-dependently, but NT-4 had little influence on the
survival rate. The current method was useful in evaluating the effects
of neuroprotective factors or neurotoxic factors on cultured
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