G, the CFSE-labeled cells were co-cultured in the 48 well-plate with the purified CD4+CD25+ T regulatory cells after with UC-MSCs education or without UC-MSCs education, at the density of 5?05/well/ml with 10 ug/ml PHA (Sigma, USA). The suspending cells were harvested and used for the flow analysis at 488 nm excitation.Cell transplantationThe purified CD4+CD25+ T regulatory cells from Tg mice spleen lymphocytes after with UC-MSCs education were administered to Tg mice (n=15) by intracardiac injection at the dose of 0.5?06 cells/100 PBS for the first time, followed by a second injection at the dose of 0.2?06 cells/100 PBS one week later. Another Tg mice (n=15) were injected the same volume of PBS as the control. Two weeks after the second injection of CD4+CD25+ T regulatory cells, the mice were performed at behavioral test.Behavioral experimentBehavioral experiment was performed on Tg mice at three weeks after the initial injection of CD4+CD25+ T regulatory cells (n=15) or PBS (n=15) and C57BL6 mice of same age (n=15) as control. Morris water maze (MWM) test was conducted to evaluate spatial memory performance in these animals. Detailed methodology is previously described by Vorhees CV [38]. In brief, the pool (1.2m diameter) was sited in a well-light room (24 ), and distinct visual cues were placed on the walls of the pool. The mice were released at four different start point (N, E, SE, NW) every day for the next consecutive 5 days toELISAThe remaining mice (n=9) were killed by over dose 10 chloral hydrate (4ml/kg weighht, i.p.). Before the mice died, the peripheral blood was harvest in the tube with heparin sodium to Fexinidazole prevent blood from agglutination. We got the plasma for testing cytokine after centrifugation after centrifugation (800 rpm/min, 5mins) and stored at -80 . The cytokine IL-10, TGF1 and IFN- were measured by ELISA kits (eBioscience, USA) according to the manufacturer’s recommendation. After the mice died, we 56-59-7 immediately removed the brain on ice andTregs Improved Impaired Cognition of ADtransferred the brains to -80 refrigerator for storage. We obtained the whole brain tissue homogenization according to according to the manufacturer’s recommendation (A1-40 A1-42, Invitrogen, USA). In brief, the whole of brain tissue was placed in a 1:8 dilution of 5M ice guanidine HCl/50mM Tis HCl and thoroughly minced. The homogenate was diluted at a ratio of 1:50 with dilution buffer (PBS with 5 BSA, 0.03 Tween-20, pH7.4) containing an inhibitor protease complex and centrifuged at 16,000 rpm for 30 min at 4 . The samples and protein standard were added t into the 96-well plate and were recorded at 450 nm using microplate reader (Biorad, USA). Each standard and experimental sample was run in duplicate and the results were averaged.Transplantation of UC-MSCs educated CD4+CD25+ T regulatory cells regulated the levels of cytokines in the plasma of APPswe/PS1dE9 transgenic miceTo examine whether CD4+CD25+ T regulatory cells after UCMSCs education could still exert immunoregulatory function in vivo, we measured the levels of plasma pro-inflammatory (interferon-) and anti-inflammatory cytokines (IL-10 and TGF1) by 23977191 ELISA kits at the end of Morris water maze. As illustrated in Figure 2A 2B, we found that the plasma levels of the cytokine TGF-1 and IL-10 were both significantly increased in the plasma of the Tg mice receiving CD4+CD25+ T regulatory cells after UC-MSCs education in vitro for 3 days compared to the Tg mice receiving PBS. I.G, the CFSE-labeled cells were co-cultured in the 48 well-plate with the purified CD4+CD25+ T regulatory cells after with UC-MSCs education or without UC-MSCs education, at the density of 5?05/well/ml with 10 ug/ml PHA (Sigma, USA). The suspending cells were harvested and used for the flow analysis at 488 nm excitation.Cell transplantationThe purified CD4+CD25+ T regulatory cells from Tg mice spleen lymphocytes after with UC-MSCs education were administered to Tg mice (n=15) by intracardiac injection at the dose of 0.5?06 cells/100 PBS for the first time, followed by a second injection at the dose of 0.2?06 cells/100 PBS one week later. Another Tg mice (n=15) were injected the same volume of PBS as the control. Two weeks after the second injection of CD4+CD25+ T regulatory cells, the mice were performed at behavioral test.Behavioral experimentBehavioral experiment was performed on Tg mice at three weeks after the initial injection of CD4+CD25+ T regulatory cells (n=15) or PBS (n=15) and C57BL6 mice of same age (n=15) as control. Morris water maze (MWM) test was conducted to evaluate spatial memory performance in these animals. Detailed methodology is previously described by Vorhees CV [38]. In brief, the pool (1.2m diameter) was sited in a well-light room (24 ), and distinct visual cues were placed on the walls of the pool. The mice were released at four different start point (N, E, SE, NW) every day for the next consecutive 5 days toELISAThe remaining mice (n=9) were killed by over dose 10 chloral hydrate (4ml/kg weighht, i.p.). Before the mice died, the peripheral blood was harvest in the tube with heparin sodium to prevent blood from agglutination. We got the plasma for testing cytokine after centrifugation after centrifugation (800 rpm/min, 5mins) and stored at -80 . The cytokine IL-10, TGF1 and IFN- were measured by ELISA kits (eBioscience, USA) according to the manufacturer’s recommendation. After the mice died, we immediately removed the brain on ice andTregs Improved Impaired Cognition of ADtransferred the brains to -80 refrigerator for storage. We obtained the whole brain tissue homogenization according to according to the manufacturer’s recommendation (A1-40 A1-42, Invitrogen, USA). In brief, the whole of brain tissue was placed in a 1:8 dilution of 5M ice guanidine HCl/50mM Tis HCl and thoroughly minced. The homogenate was diluted at a ratio of 1:50 with dilution buffer (PBS with 5 BSA, 0.03 Tween-20, pH7.4) containing an inhibitor protease complex and centrifuged at 16,000 rpm for 30 min at 4 . The samples and protein standard were added t into the 96-well plate and were recorded at 450 nm using microplate reader (Biorad, USA). Each standard and experimental sample was run in duplicate and the results were averaged.Transplantation of UC-MSCs educated CD4+CD25+ T regulatory cells regulated the levels of cytokines in the plasma of APPswe/PS1dE9 transgenic miceTo examine whether CD4+CD25+ T regulatory cells after UCMSCs education could still exert immunoregulatory function in vivo, we measured the levels of plasma pro-inflammatory (interferon-) and anti-inflammatory cytokines (IL-10 and TGF1) by 23977191 ELISA kits at the end of Morris water maze. As illustrated in Figure 2A 2B, we found that the plasma levels of the cytokine TGF-1 and IL-10 were both significantly increased in the plasma of the Tg mice receiving CD4+CD25+ T regulatory cells after UC-MSCs education in vitro for 3 days compared to the Tg mice receiving PBS. I.
