Mic approach. Although the analytical variance of the PBMC fraction using 2D gel electrophoresis within and between laboratories is already described [14], we use the 2D-DIGE approach, which is more common nowadays for differential proteomic analysis. Moreover, we focus in this study on theVariation in PBMC Proteomeinterindividual variance as the proteome of 24 elderly healthy volunteers is compared. We will also establish the percentage of high variable proteins and look into the factors that contribute to the technical variation in our experiments. Next, the sample size for experiments using a 2D-DIGE approach with PBMC fractions is determined. This way, an appropriate setup can be proposed for future 2D-DIGE discovery experiments using the PBMCs.Materials and Methods Ethics statementThe blood samples were taken with the approval of the local ethical committee (Clinical Trial Center, UZ Leuven, Campus Gasthuisberg, ML6505) and a signed informed consent from every volunteer is available.PBMC samplingBlood from 24 healthy volunteers (15 males, 9 females, ages 65?86, with no clinical signs of inflammation)(Table 1) was 
collected in 461.8 ml 0,109 M buffered sodium citrate vacutainers (Venosafe, VWR, Leuven, Belgium) and were processed within 4 hours after blood withdrawal. For the isolation of the PBMC cells, leucosep tubes (Greiner Bio-One, Wemmel, Belgium) were used. Blood was diluted 1:1 with Dulbecco’s Phosphate Buffered Saline (PBS)(Sigma, St Louis, Missouri) prior to transferring it into the leucosep tube. After centrifugation (10 min, 1000 g and ambient temperature), the PBMC cell layer of two leucosep tubes were pooled and transferred into a 15 ml falcon. To wash the PBMCs, the sample was diluted with 10 ml PBS and centrifuged again for 10 min at 250 g and ambient temperature. The obtained cell pellet was resuspended in 10 ml PBS, to wash the cells a AN-3199 cost second time. After centrifugation (10 min, 250 g, ambient temperature), the cell pellet was stored at 280uC until further analysis.equilibrated by soaking them in sodium dodecyl sulphate (SDS) equilibration buffer (50 mM tris Cl pH 8.8, 6 M urea, 30 glycerol, 2 SDS) with 1 DTT. After 15 min, the strips were placed in SDS equilibration buffer with 4 iodoacetamide and bromophenolblue, which was added as tracking dye. Spotpickgels were created by using Bind-Saline working solution (12 ml ethanol, 300 ml acetic acid, 15 ml bind-silane, 2.7 ml MilliQ) to stick the gel to the plate. The strips were then added on the SDS-PAGE gels (12 ), and covered with agarose. The second dimension was carried out with an Ettan DIGE Twelve electrophoresis system (GE Healthcare) with following parameters: 600 V, 8 mA/gel and 10 v. After one hour, the settings were changed to 12 mA/gel. The second dimension was stopped after 20 h and the gels were scanned using the Ettan DIGE imager. The resolution was set at 100 mm, and the get Calciferol scanning exposure time was optimized for every gel, to prevent saturation of interesting protein spots. After scanning the gel, total protein content was visualized by Deep purple staining. In short, gels were fixated overnight in 10 methanol, 7.5 acetic acid. The next day, the gels were washed and then stained with deep purple dye for 1 h. After 2 washing steps with 7.5 acetic acid, the gels were scanned at 590 nm.Image analysisThe gel images were loaded into the Decyder 2D 7.0 software (GE Healthcare). In the Differential In Gel Analysis module, settings for optimal intra-gel s.Mic approach. Although the analytical variance of the PBMC fraction using 2D gel electrophoresis within and between laboratories is already described [14], we use the 2D-DIGE approach, which is more common nowadays for differential proteomic analysis. Moreover, we focus in this study on theVariation in PBMC Proteomeinterindividual variance as the proteome of 24 elderly healthy volunteers is compared. We will also establish the percentage of high variable proteins and look into the factors that contribute to the technical variation in our experiments. Next, the sample size for experiments using a 2D-DIGE approach with PBMC fractions is determined. This way, an appropriate setup can be proposed for future 2D-DIGE discovery experiments using the PBMCs.Materials and Methods Ethics statementThe blood samples were taken with the approval of the local ethical committee (Clinical Trial Center, UZ Leuven, Campus Gasthuisberg, ML6505) and a signed informed consent from every volunteer is available.PBMC samplingBlood from 24 healthy volunteers (15 males, 9 females, ages 65?86, with no clinical signs of inflammation)(Table 1) was collected in 461.8 ml 0,109 M buffered sodium citrate vacutainers (Venosafe, VWR, Leuven, Belgium) and were processed within 4 hours after blood withdrawal. For the isolation of the PBMC cells, leucosep tubes (Greiner Bio-One, Wemmel, Belgium) were used. Blood was diluted 1:1 with Dulbecco’s Phosphate Buffered Saline (PBS)(Sigma, St Louis, Missouri) prior to transferring it into the leucosep tube. After centrifugation (10 min, 1000 g and ambient temperature), the PBMC cell layer of two leucosep tubes were pooled and transferred into a 15 ml falcon. To wash the PBMCs, the sample was diluted with 10 ml PBS and centrifuged again for 10 min at 250 g and ambient temperature. The obtained cell pellet was resuspended in 10 ml PBS, to wash the cells a second time. After centrifugation (10 min, 250 g, ambient temperature), the cell pellet was stored at 280uC until further analysis.equilibrated by soaking them in sodium dodecyl sulphate (SDS) equilibration buffer (50 mM tris Cl pH 8.8, 6 M urea, 30 glycerol, 2 SDS) with 1 DTT. After 15 min, the strips were placed in SDS equilibration buffer with 4 iodoacetamide and bromophenolblue, which was added as tracking dye. Spotpickgels were created by using Bind-Saline working solution (12 ml ethanol, 300 ml 
acetic acid, 15 ml bind-silane, 2.7 ml MilliQ) to stick the gel to the plate. The strips were then added on the SDS-PAGE gels (12 ), and covered with agarose. The second dimension was carried out with an Ettan DIGE Twelve electrophoresis system (GE Healthcare) with following parameters: 600 V, 8 mA/gel and 10 v. After one hour, the settings were changed to 12 mA/gel. The second dimension was stopped after 20 h and the gels were scanned using the Ettan DIGE imager. The resolution was set at 100 mm, and the scanning exposure time was optimized for every gel, to prevent saturation of interesting protein spots. After scanning the gel, total protein content was visualized by Deep purple staining. In short, gels were fixated overnight in 10 methanol, 7.5 acetic acid. The next day, the gels were washed and then stained with deep purple dye for 1 h. After 2 washing steps with 7.5 acetic acid, the gels were scanned at 590 nm.Image analysisThe gel images were loaded into the Decyder 2D 7.0 software (GE Healthcare). In the Differential In Gel Analysis module, settings for optimal intra-gel s.
