Antibody stock solutions: Master Stocks and Working Stocks
We are concerned with two types of stock solutions of antibodies for flow cytometry:
Master stocks
These are the stocks produced after the labeling reactions. They are generally at concentrations as low as 0.2 mg/ml and as high as 4 mg/ml. Master stocks should be carefully stored in sterile conditions, at a minimum with sodium azide to prevent microbial growth. Even better, it might be desireable to sterile filter the master stocks, and to open them only in a biosafety cabinet.
The optimal staining titer of the antibody should be determined with by performing a serial dilusiton series with this stock. This should be done carefully, or else the preparation of the working stocks will not translate.
Working stocks
Working stocks are diluted to concentration convenient for pipetting into stains. This turns out to be a compromise between:
- Having the antibodies concentrated such that the volume of antibody added to a stain is negligible compared to the total volume of the stain (e.g. 1 µl of antibody per 100 µl of cells).
- Having the antibody dilute enough so that the required volume can be accurately pipetted and waste is minimized (e.g. 5 µl of antibody per 100 µl of cells).
In the Altman lab standard, the standard working antibody stock is 20x, corresponding to 5 µl for a 100 µl test.
Working stocks should be prepared in Tris buffered saline (10 mM Tris, 150 mM NaCl, pH 8.2) in the presence of sodium azide (0.05%) and a protein stabilizer. Appropriate protein stabilizers are:
- Protease free BSA, at a concentration of 1-2% (10-20 mg/ml)
- Gelatin (0.1%)
Test for reproduction of titering data
Data obtained from use of the “working stocks” should qualitatively match that obtained from the initial titration of the master stocks. This depends upon the accuracy of the titration of the master stock, as well as the preparation of the working stock.
Test for stability
The working stocks should be tested after one month and six months to determine stability