BMCMC degranulation assay (ß-hexosaminidase release) (PDF)

	BMCMC in culture (10⁶ cells)
	96 well V-bottom and flat-bottom plates
	Tyrode’s Buffer*:	10 ml 1M HEPES 7.54 g NaCl 0.37 g KCl 0.206 g CaCl₂ 0.203 g MgCl₂ 1.008 g Glucose 1 g BSA
		*bring up to 1L with dH₂O and filter through 0.2mm filter. Store at 4°C.
	p-NAG (substrate)**:	buffer is 14.29 g Na₂HPO₄, 42.05 g citric acid brought up to 500 ml, pH 4.5
		**Prepare 1.3mg/ml (4mM) p-NAG (p-Nitrophenyl-N-acetyl-ß-D-glucosaminidine) in substrate buffer by vortexing vigorously and/or stirring with heating plate. Store 10 ml aliquots at -20°C.
	0.2M glycine:	1.5 g glycine brought up to 100 ml, pH 10.7 (use NaOH), stored at room temperature.

Preload 10⁶ BMCMC in normal BMCMC medium plus 2 µg/ml IgE (α-DNP) at 37°C for 16-18 hours.
Wash cells 1X Tyrode’s buffer to remove unbound IgE by spinning down cells at 1000 RPM (Beckman Allegra 6R Centrifuge) for 5 minutes and resuspending in an equal volume of Tyrode’s to wash.
Spin down cells and resuspend in 160 µl Tyrode’s buffer.

Transfer 10µl cells + 10 µl 2X stimulus to 96 well V-bottom plate (each condition in duplicate).

2X stimulus:

2000ng/ml DNP
200ng/ml DNP
20ng/ml DNP
2ng/ml DNP
0ng/ml DNP (Tyrode’s)
50ng/ml PMA + 10µM A23187

Incubate plate at 37°C for 60’. (Thaw substrate at 37°C during this step!)
Centrifuge plate at 1000 RPM for 5 minutes.
Transfer 10 µl supernatant to a 96 well flat-bottom plate to save. Remove the remainder of supernatant (7-10 µl) from each well and discard these supernatants.
Add 20 µl 0.5% Triton-X (made up in Tyrode’s) to each pellet. Pipette up and down to lyse cells.
Transfer 10 µl of the pellet lysate to a second 96 well flat-bottom plate.
Add 50 µl p-NAG (substrate) to each well containing supernatant from step 7 or pellet lysate from step 9. Incubate plate at 37°C for 60 minutes.
Add 150 µl 0.2M glycine, pH10.7 to each well.
Read plate at 405 nm in a 96 well plate reader.