4 replies to this topic

[ollorin]

Hello everyone,

 

I have to work on a protocol to isolate epithelial cells but I have a problem with the formulation of a media.

 

« Culture medium : (Collection media with 5 % FCS, 120IU/ml of Insulin) »

Collection media is previously defined as a mix of DMEM/Ham's F12 and antibiotics, nothing unusual and I don't need a lot of culture medium (100-200mL for the whole protocol).

 

I just have a problem with the amount of Insulin (normally, IU means unit of insulin, not international unit). I use Insulin from Sigma :

http://www.sigmaaldr...ng=en&region=GB

 

This Insulin has an activity of 27.5 IU/mg (which is good). The solubility of insulin in water and ethanol is low, 10mg/mL in water : 275 IU/mL for a 10mg/mL solution.

I need a media with 120 IU, which means that 43 % of my final volume comes from the Insulin solution...that's too much.

Maybe I did a mistake or i misunderstood something (units?) but due to the low solubility I don't know (and I didn't find) how to prepare a higher stock solution or an other way to add Insulin?

 

Thank you in advance for your help,

Have a nice day!

Edited by ollorin, 29 December 2014 - 02:49 AM.

[bob1]

120 IU total or per ml? If the first, then just add the required fraction of a ml to your medium. The other way to do it is to dissolve the insulin in the medium and then sterile filter.

[ollorin]

It's 120 IU per mL, which is a high concentration.

 

I thought to dissolve insulin powder in my medium and to sterilize it with filter too, but in general i prefer not to filter media with hormones (there is alway a loss of a few drop in the filter).

 

Anyway thank you for answering and i guess that if there is no alternative I'll have to add directly my powder .

[bob1]

Some protocols say to use low pH for dissolving insulin, just google for dissolve insulin and see what you get. 120 IU per ml is quite a lot I think, are you sure that is the right concentration?

[ollorin]

Yes according to Sigma recommandations, it's better to dissolve Insulin at low pH. However I can't prepare this kind of solution and to add it in my media, the volume is too important if we keep a stock solution of 10mg/mL of Insulin as recommanded.

 

Yes this is the rigth concentration. This is the source :

http://www.sciencedi...569199304000657

 

2. Materials and methods

2.2. Isolation and culture of murine tracheal epithelial cells

 

... Epithelial cells were dissociated by gentle agitation, pooled, washed, and incubated in Culture medium (Collection media with 5% FCS, 120 IU/ml of Insulin) at 37 °C for 2 h ...

Edited by ollorin, 30 December 2014 - 02:29 AM.