For analysis of apoptosis of [CD115.sup.+] monocytes in vitro, bone marrow [CD115.sup.+] monocytes (1 x [10.sup.6]/mL) were seeded in 6 cm plates, cultured overnight, and then incubated with different concentrations (20, 50, 150, 300, 500, and 800 [micro]g/ mL) of AGEs (purified AGE-BSA
, Anyan-bio Technology, Shanghai, China, catalog number AY4710P) for 24 h; BSA (Sigma-Aldrich, Missouri, USA, catalog number A1933) was used as the control vehicle.
The number of immunopositive cells in retinas cultured in AGE-BSA supplemented with citicoline was significantly fewer than that in glucose-AGE-BSA without citicoline (37.3 [+ or -] 5.6% versus 46.5 [+ or -] 5.8%; P = 0.0011; Figures 2 and 3).
The number of caspase-9-positive cells in retinas cultured in AGE-BSA supplemented with citicoline was significantly fewer than that in glucose-AGE-BSA without citicoline (25.9 [+ or -] 5.1% versus 58.0 [+ or -] 9.8%; P < 0.0001; Figures 4 and 5).
In AGEs-exposed retinas supplemented with NT-4 media (glucose-AGE-BSA + NT-4) (g), in the triplet media (glucose-AGE-BSA + citicoline + TUDCA + NT-4) (h), in the doublet media (glucose-AGE-BSA + citicoline + TUDCA) (f), in citicoline media (glucose-AGE-BSA + citicoline) (d), in TUDCA media (glucose- AGE-BSA + TUDCA) (e), and in RAGE inhibitor media (glucose-AGE-BSA + RAGE-I) (c), the number of caspase-9-immunopositive cells is fewer than that in AGEs-exposed retinas without the neurotrophic factors.
Horseradish peroxidase (HRP)-labeled AGE-BSA (HRP-AGEBSA, Peroxidase Labeling Kit-NH2; Dojindo, Japan) and serially diluted OSSC extracts were incubated for 2h at room temperature.
Cells were grown to 80% confluency in 8-well slides, synchronized, and exposed for 24 h to AGE-BSA (100 [micro]g/ml) in the absence or presence of 100 ng/ml OSSC extracts.
PDL fibroblasts were randomly divided into 3 groups: AGE-BSA (group A), BSA (group B), and control (group C).
To determine the extent of RAGE expression after AGE-BSA treatment, human PDL fibroblasts were subjected to AGE-BSA (group A), BSA (group B), or given no treatment (control group C).
The method of Bradford  was used for quantification of proteins (BSA and AGE-BSA after dialysis).
The most pronounced difference between the relative fluorescence ([[lambda].sub.em] = 440 nm) of albumin (BSA) and AGE-BSA was seen with excitation at 247 nm (Fig.
Klotho Overexpression Protected PTECs from Injury Induced by AGE-BSA. Next, we further demonstrated the critical role of klotho on PTEC injury after exposure to AGE-BSA by upregulating klotho expression.
More interestingly, in addition to attenuating the TEC injury induced by AGE-BSA, klotho overexpression increased the cellular resistance to [H.sub.2][O.sub.2] insult, indicating that klotho's renoprotection was partially through antioxidative action which agreed with previous studies .