Contributor: Suprya Jayadev
Date: January 10, 1991
NBT solution
1.5 mls - PBS
3 mg - NBT powder
3 µl - 0.162 mM TPA (stock 1 mg/ml; 1.62 mM)
Procedure
1) Spin ~1 ml of cells at 1500 rpm for 5 mins.
2) Resuspend pellet in 100 µl PBS + 100 µl NBT solution.
3) Incubate in 370C water bath for 1 hour.
4) Stop reaction with 3 ml ice cold PBS.
5) Spin at 1500 rpm for 5 mins.
6) Resuspend in 200 µl PBS and load 100 µl into slide chamber.
7) Spin at 3000 rpm for ~1 min.
8) Allow slides to dry.
9) Fix with methanol - 1 minute.
10) Rinse with water.
11) Counterstain with Safranin-O - 1.5 mins.
12) Rinse off excess stain.
13) Allow slides to dry and count cells.