MEDIA FORMULATION:

S.C. (“Supplementum Completum”)

Goals and Rationale:

An additive to Tissue Culture media, providing antibiotics, glutamine, 2ME and HEPES. Formulated for RPMI 1640, but S.C. also works for other media, including EHAA, DMEM (may need to supplement with pyruvate) and IMDM.

 

Safety Concerns

         Caution: This protocol uses 2-Mercaptoethanol - Follow the specific handling and disposal procedures.

 

Method

To make up 1 Liter of S.C., as a 20X stock

 

     Chemical                            Amount       [Stock] (20x)         [Final] (in media)

 

1)  HEPES                                23.83g          100mM                  5mM

     Life Technologies #11344-033 [powder; Mol.Wt. = 238.3].

2)  Penicillin/Streptomycin       200ml           1,000U(mg)/ml        50U(mg)/ml

     Life Technologies #15070-022 [100ml in saline: 5,000 U/ml (Pen), 5mg/ml (Strep)]

     (From UM Stores #CX30311).

3)  L-Glutamine                        6.0g             41mM                   2mM

     Life Technologies #21051-024 - [powder, Mol.Wt. = 146.1]

4)  2-Mercaptoethanol              70ml             1mM                     50mM

     Sigma #M-7522 [liquid, “straight” concentration = 14.3M]

5) Gentamicin Sulfate              20ml            1mg/ml                 50mg/ml

     Irvine Scientific #9355 [20ml in saline, 50mg/ml)

 

Ø     Dissolve all dry products in 500ml of RPMI 1640 (Life Technologies #21879-076)*.

Ø     Add liquid components.

Ø     Adjust volume* to 1L with RPMI 1640.

Ø     Sterile filter (0.2mm) and store in 25ml aliquots at -20^oC.

 

     Use at 5%, i.e. 25ml/ 500ml media.

 

*Remember to use tissue culture grade plastic/glassware for preparing these solutions.