PI/RNase Staining Buffer

Protocol tips

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- Cells were were incubated with various concentrations of 1 (0, 25, 50, 75, 100, 150 and 200 µM); 2 (200 µM); 3 (200 µM); 4 (200 µM); 5 (500 µM); a mixture of ManNAc (125 µM) and 5 (500 µM); and ManNAc (10 mM) for five days.	- Fixed with 78% aqueous ethanol and incubated for 24h

Upstream tips
- Cells were were incubated with various concentrations of 1 (0, 25, 50, 75, 100, 150 and 200 µM); 2 (200 µM); 3 (200 µM); 4 (200 µM); 5 (500 µM); a mixture of ManNAc (125 µM) and 5 (500 µM); and ManNAc (10 mM) for five days.
Protocol tips
- Fixed with 78% aqueous ethanol and incubated for 24h

Publication protocol

Cell cycle arrest was studied following an established protocol [4]. Briefly, Jurkat cells (2.3 × 105 cells/mL) were incubated with various concentrations of 1 (0, 25, 50, 75, 100, 150 and 200 µM); 2 (200 µM); 3 (200 µM); 4 (200 µM); 5 (500 µM); a mixture of ManNAc (125 µM) and 5 (500 µM); and ManNAc (10 mM) for five days. The cells were then harvested, counted, washed twice with PBS, re-suspended in PBS (500 µL), added to a cold solution of 78% aqueous ethanol (4.5 mL) using a glass Pasteur pipette and kept at 2.0°C for at least 24 h. The fixed cells were centrifuged twice to remove the ethanol completely, re-suspended in propidium iodide (PI) / ribonuclease A (RNase A) staining buffer (BD Pharmingen, Catalog No. 550825, San Diego, CA) (500 µL for 1.0 × 106 cells), incubated at 37°C for 15 min and analyzed by flow cytometry. The cellular aggregates were excluded from single cell populations using an area-width plot (FL2 – PI fluorescence) for the cell cycle status determination. At least 10,000 gated events were counted for each determination (Figures 4 and 6G in main text).

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