Start with at least 1 × 106 cells per condition, to obtain at least 200,000 events analysed by flow cytometry at the end of the protocol. Conditions should include: the test samples (treated with kynurenine); background fluorescence control samples (matched samples, not treated with kynurenine, thus allowing for identification of cells exhibiting fluorescence above background); specificity controls such as System L blocked samples (BCH-treated, or Leu-treated), or uptake performed on ice (4 °C) (to determine transported kynurenine as opposed to surface binding); positive controls such as cell treatments driving high expression of System L transport. If required, surface cell antibody staining should be performed prior to uptake assay protocol. This may be performed at room temperature or 4 °C, however test samples must be warmed to 37 °C, e.g. in water bath, prior to kynurenine uptake. (As with all multi-parameter flow cytometry, appropriate antibody staining controls must also be performed.) Samples can be fixed immediately after uptake assay by addition of 4% (vol/vol) paraformaldehyde (PFA; to a final concentration of 1%) for 30 min at room temperature.
For kynurenine uptake assay; pre-warm kynurenine (800 μM, in HBSS), BCH (40 mM, in HBSS) and lysine (20 mM, HBSS) and HBSS to 37 °C. After surface antibody staining of samples, resuspend cells in 200 μl warmed HBSS (1–5 × 106 cells in FACS tubes, or scale accordingly into plates). Keep cells in water bath at 37 °C. Add 100 μl of HBSS, or BCH or lysine to appropriate samples. Add 100 μl HBSS to no kynurenine controls (final volume 400 μl). Finally, add 100 μl kynurenine to appropriate samples. Stop uptake after 4 min by adding 125 μl 4% PFA for 30 min at room temperature, in the dark. The final concentrations for uptake assay are: 200 μM kynurenine; +/-10 mM BCH; +/-5 mM lysine. After fixation, wash cells twice in PBS/0.5% BSA and resuspend in PBS/0.5% BSA prior to acquisition on flow cytometer. The 405 nm laser and 450/50 BP filter are used for kynurenine fluorescence detection.
To monitor kynurenine uptake in live cells, acquire data on flow cytometer immediately following addition of kynurenine and plot fluorescence against time.
For kynurenine uptake assay; pre-warm kynurenine (800 μM, in HBSS), BCH (40 mM, in HBSS) and lysine (20 mM, HBSS) and HBSS to 37 °C. After surface antibody staining of samples, resuspend cells in 200 μl warmed HBSS (1–5 × 106 cells in FACS tubes, or scale accordingly into plates). Keep cells in water bath at 37 °C. Add 100 μl of HBSS, or BCH or lysine to appropriate samples. Add 100 μl HBSS to no kynurenine controls (final volume 400 μl). Finally, add 100 μl kynurenine to appropriate samples. Stop uptake after 4 min by adding 125 μl 4% PFA for 30 min at room temperature, in the dark. The final concentrations for uptake assay are: 200 μM kynurenine; +/-10 mM BCH; +/-5 mM lysine. After fixation, wash cells twice in PBS/0.5% BSA and resuspend in PBS/0.5% BSA prior to acquisition on flow cytometer. The 405 nm laser and 450/50 BP filter are used for kynurenine fluorescence detection.
To monitor kynurenine uptake in live cells, acquire data on flow cytometer immediately following addition of kynurenine and plot fluorescence against time.
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