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. 2009 Jan 1;340(1):11–24. doi: 10.1016/j.jim.2008.09.014

Fig. 1.

Fig. 1

Dasatinib substantially improves pMHC tetramer staining intensity. A. 105 ILA1 CTL were re-suspended in 40μl of PBS ± 50 nM dasatinib or Lck inhibitor II (Calbiochem), then incubated at 37 °C for 30 min. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10µg/ml for 20 min at 37 °C, washed twice in PBS and analyzed on a FACSCalibur (BD) flow cytometer. A > 10-fold increase in median fluorescence intensity (MFI) was observed after treatment with 50 nM dasatinib (blue) or LcK inhibitor II (red) compared to staining without PKI pre-treatment (green line). B. 105 ILA1 CTL were treated with various concentrations of dasatinib for 30 min at 37 °C, then stained with either HLA A2/ILAKFLHWL tetramer or the non-cognate HLA A2/ELAGIGILTV tetramer for 20 min at 37 °C before washing with PBS. C. 105 ILA1 CTL were resuspended in 40μl of PBS ± the indicated concentration of dasatinib and incubated for 60 min at 37 °C. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10 μg/ml for 20 min at 37 °C and washed twice in PBS prior to flow cytometric analysis. D. As (A), but ILA1 CTL were incubated with 50 nM dasatinib for various times prior to staining with pMHCI tetramer. For this experiment the drug was washed off prior to staining. E. As (A), but tetramer concentration was varied to stain CTL pre-treated ± 50 nM dasatinib for 30 min. F. 105 Mel13 CTL were stained with various concentrations of HLA A2/ELAGIGILTV tetramer following incubation ± 50 nM dasatinib for 30 min. G. 5x105 splenocytes from an F5 TCR transgenic Rag+ mouse were resuspended in PBS ± 50 nM dasatinib and incubated for 30 min at 37 °C. Cells were subsequently stained with H2-Db/ASNENMDAM-PE tetramer for 20 min at 37 °C followed by anti-CD8 Cy5.5 for 30 min on ice prior to two washes in PBS and analysis by flow cytometry. H. 105 cells of the HLA DR⁎0101-restricted, influenza virus A HA307-319 PKYVKQNTLKLAT-specific CD4+ clone C6 were incubated with PBS ± 50 nM dasatinib for 30 min at 37 °C, then stained with cognate PE-conjugated tetramer for 20 min at 37 °C. Samples were washed with PBS before flow cytometric analysis. Irrelevant tetramer was used as a negative control in all cases.