Reagent	Cat Number	FW	Final	For 500 ml
1	Iscove’s MDM, liquid *	Gibco #12440-053	1X Liquid	1X	500 ml bottle
2	Hypoxanthine	Sigma #H9377	136.1 g/mol	1 mM	68 mg
3	Bathocuproine Disulfonate	Sigma #B125	564.5 g/mol	50 uM	14.1 mg
4	L-Cysteine • H2O	Fisher BP376-100	175.6 g/mol	1.5 mM	91 mg
5	2-mercaptoethanol	Sigma #M6250	14.3 M	0.2 mM	7.2 ul
6	Thymidine	Sigma #T9250	242.2 g/mol	0.16 mM	19.5 mg
7	Na-Pyruvate	Gibco #11360-070	100 mM	1 mM	5 ml
8	Pen/Strep (+ 29.2 mg/ml L-Gln) Stored at –20, thaw at 37 deg and invert several times to resuspend	Gibco #10378-016	10,000 u/ml	100 u/ml	5 ml

1. Dissolve all reagents in 500 ml of pre-warmed (37 deg C) Iscove’s MDM by stirring at RT. This can be done right in the Iscove’s MDM bottle. Be sure all powders are dissolved.

2. If necessary, adjust pH to 7.4 with 1N NaOH

3. Filter Sterilize (0.22 um filter unit) and store at 2 – 8 deg C.

4. Add Heat-Inactivated Fetal Calf Serum to 15 % prior to use.