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NBT assay: Brice Weinberg protocol
Contributor: Suprya Jayadev
Date: January 10, 1991
NBT solution
3 mg. - NBT
10 µl - 20 µg/ml PMA
1.5 ml - RPMI 1640 (incomplete)
Procedure
1) Take out ~1.0 X 106 cells total and wash one time with PBS in a cloudy
5 ml Rohen tube.
2) Resuspend pellet in 1 ml NBT solution. (Resuspend well!!)
3) Incubate at 370 C for 15 mins.
4) Wash with 1 ml cold PBS - repeat for a total of 3X.
--> Spin at 1500 rpm for 5 mins. each time.
5) Resuspend final pellet in 200 µl PBS and load 100 µl into slide
well.
6) Spin at 3000 rpm for ~1 min.
7) Allow slide to dry.
8) Fix with methanol - 1 min.
9) Rinse in water.
10) Counterstain with 0.15% Safranin O: 30 sec. - 1 min.
11) Rinse off excess stain.
12) Allow slide to dry and count.