Table 1.

HDAC-GLO Deacetylase Analysis. (A) Descriptive Calculation and Explanation of Compound Dilutions to Achieve a 100 nm–25 µm Treatment Range. Sample Plate Layout (96-Well Format) for Each Compound in Triplicates [Boxes 1, 2, 3 for Tubastatin A (Tuba A) and 4, 5, 6 for Entinostat]. (B) Descriptive Calculations for HDAC-GLO Media Mix Preparation

(A) Compound stock: Make enough for 10 wells for each compound (Note: for each compound prepare the stock in tube 1 of the 8-well strip). Stock concentration for each compound should be adjusted to 25 µM
Add media to tubes 2–8 (as calculated in 2nd and 5th columns) and to dilute the stock as indicated
	Tuba A			Entinostat
1	25 µM stock		1	25 µM stock
2	90 µL media	60 µL from tube 1	2	90 µL media	60 µL from tube 1
3	75 µL media	75 µL from tube 2	3	75 µL media	75 µL from tube 2
4	75 µL media	75 µL from tube 3	4	75 µL media	75 µL from tube 3
5	135 µL media	15 µL from tube 2	5	135 µL media	15 µL from tube 2
6	135 µL media	15 µL from tube 3	6	135 µL media	15 µL from tube 3
7	135 µL media	15 µL from tube 5	7	135 µL media	15 µL from tube 5
8	DMSO	2.5 µL DMSO in 147.5 µL media	8	Triton X	1.5 µL of Triton X in 148.5µL media

(B) Final 96-Well Plate Layout (in Triplicates)
	Tuba A		Entinostat			Additional Compounds			Additional Compounds
	1	2	3	4	5	6	7	8	9	10	11	12
A		25,000			25,000			25,000			25,000
B		10,000			10,000			10,000			10,000
C		5000			5000			5000			5000
D		2500			2500			2500			2500
E		1000			1000			1000			1000
F		500			500			500			500
G		100			100			100			100
H	DMSO control (Neg)			Triton X control (Pos)			Untreated control

Preparation of HDAC-GLO media mix. 1-Premix HDAC-GLO substrate + reagent (included in the Promega kit) and aliquot into 1500 µL volume.

To add 5 µL of developer (included in Promega kit) in 1500 µL premix HDAC-GLO solution.