2-D gel electrophoresis was performed using immobilized pH gradient stripes (BioRad ReadyStrip™ IPG Stripes, pH 4–7, 17 cm). L-plastin was detected on western blots and quantified using a densitometer as described elsewhere 8. The phosphorylation was calculated as percent phosphorylated L-plastin by dividing the grey value of phosphorylated
L-plastin (right spot) by the grey value of total L-plastin (sum the grey values of both spots). PB T cells were stimulated with crosslinked Abs as indicated, washed once with PBS/0.5% FCS, and fixed in 75% v/v ethanol. Fixed cells were preserved o/n at 4°C and afterward washed with PBS/0.5% FCS and stained for 30 min at room temperature using 20 g/mL PI, 100 g/mL RNase A (Sigma, boiled for 15 min to inactivate DNase), and FACS buffer with 0.1% Triton X-100. Cell-cycle entry was determined according to the DNA this website content using FACSCalibur in which doublets were gated out using the width function. For the measurements of the selleck chemicals expression of surface receptors, 1×106 T cells were stained with the respective fluorescently labeled Abs. Briefly, cells were incubated with the Abs (concentration according to the manufacturer’s suggestions) in PBS (0.5% BSA, 0.07% NaN3) for 15 min at 4°C.
Thereafter, cells were washed and subjected to flow cytometry. The data acquisition was performed using a FACSCalibur and data were analyzed using CellQuestPro 8, 29, 48. For sorting of EGFP-positive cells, two
samples of 5×106 cells were used for the transfections. Cells were incubated for 24 h to express the cDNA-encoded proteins and then sorted for EGFP-positive cells using a FACS Vantage in the purity mode. The sorted cells (2×105) were immediately lysed using TKM lysis buffer and subjected to 1-D western blots. PB T cells were stimulated with crosslinked Abs and incubated at 37°C for the indicated time points. Later, cells were rinsed and stained with 2.5 μg/mL PI. Cell death was afterward analyzed using a FACSCalibur or an LSR2 (BD Bioscience). All statistical evaluations were performed using Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Groups were compared with Student’s paired t-test and considered to be statistically relevant if the p-value was CHIR-99021 below 0.05. This work was supported by the Deutsche Forschungsgemeinschaft (SA393/3 to Y. S.). The authors thank Dieter Stefan for the cell sorting. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere.