Protocols

The protocols below reported are just an example: please check with the researchers of the unit of analysis if your methods and protocols used for handling, collection, preparation and storage of samples are suitable for Luminex analysis (including volumes, concentrations, freezing/thawing, security procedures and so on).

IMPORTANT: the conditioned medium should be prepared using not higher than 0.5% of serum. If higher, please let us know.

1. Collect CM and keep on ice;
2. add protease and/or phophatase inhibitors, depending on the experimental design;
3. Centrifuge collected CM under the conditions suitable for each cell type to remove cell debris and large molecular aggregates;
4. evaluate protein concentration by Bradford’s protocol: in any case, save the data regarding the number of cells plated for the experiment;
5. if required, it is possible to ultracentrifuge CM to reduce the volume from 10 to 20 folds using a microporous membrane (e.g. 3 kDa cut off);
6. store CM at -80° C: the best option is to prepare both aliquots of 1-2 or 5 ml and 100-200 µl (suitable also for the determination of total protein concentration);
7. Provide a control sample (blank: culture medium + protease and phophatase inhibitors if added, about 1 ml);

For any question please contact the Unit.

Protocol for preparation, handling and storage of biological samples (serum, plasma or others).

There are several good protocols to prepare high quality serum or plasma samples. Since biological samples may be obtained by using different protocols often already and routinely used in clinical or research laboratories, and such protocols cannot be modified by the user, please contact the researchers working in the unit to verify whether protocols and plasticware used for vein puncture, blood withdrawal, preparation and storage are suitable.

A few simple tips:

1. avoid freeze – thaw cycles;
2. use sterilized plasticware;
3. please make small aliquots (max 100 – 200 µl);
4. use -80 °C freezers only, if possible.