Thursday July 02, 2009
Let’s face it, cell separation can be tricky. For me, it was especially mysterious. I am a graduate student in a protein signaling lab. I am working to establish a mouse model to study our key proteins. Our lab primarily works with cell lines, thus very little guidance could be offered as far as cell separation. Needless to say, I fumbled through a couple of experiments. As we all know, the protocols are usually clear and to the point; however, small handling techniques can easily make or break an assay.
I used cell enrichment kits to purify T Cells from splenocytes and/or mesynteric lymph nodes for use in apoptosis assays, stimulation assays, flow cytometry, RNA isolation, protein extraction, and various other functional assays. Consistency in yield is of the utmost importance.
I was in the midst of a cell separation breakdown when the StemCell Technologies representative stopped by my lab. This representative literally saved my experiment. He quickly jotted my information down and had a sample kit and magnet to my lab the very next day. I completed my experiment with lower than expected efficiency but with an acceptable yield. When I told my StemCell representative that my yield was low he shipped another sample kit out. The next experiment was perfection.
The EasySep Mouse CD4+ T Cell Enrichment Kit from Stem Cell Technologies is comes with the typical negative selection cocktail of antibodies against CD11b, CD19, CD45R, Cd49b, TER119. A biotin selection cocktail for conjugation to the magnetic dextran nanoparticles is also included in the kit. Lastly, the kit contains rat serum to prevent non-specific interactions. The extra supplies needed are PBS, FBS, 5mL (12 x75 mm) polystyrene tubes and magnet. The protocol for the kit is extremely clear and calls for three fifteen minutes incubations before you are ready to separate. The actual separation is quick. The separation is done in suspension not in a column, you just slide your tube into the magnet and wait five minutes and pour off the T Cell enriched supernatant. That is it. The labeled cells stick to the wall of the tube. I have used the labeled fraction for RNA isolation but not yet for functional studies.
As compared to other cell separation systems, I have found this system to be more time efficient and effective. The labeling process is about the same as other systems, however the pour off seems to be more gentle, and faster than column separation. Furthermore, the ease of the separation gives a consistent yield as far as cell numbers. For example, I usually expect 10-20% CD4+ in my splenocytes as shown by flow cytometry analysis. I usually return 16-19% in my enrichment using the EasySep Kit. My purity has never been lower than 89%.
The only negative aspect to this system is that it is for the enrichment of T Cells from spleen: other tissues may or may not work. Also, the purity maybe off if the pour-off step is done forcefully, so be careful.
Overall, the EasySep enrichment kit is truly easy separation with consistent yield. The performance of this T Cell enrichment kit combined with great customer service makes this cell separation system a worry free product!
Tiffanny Newman
Graduate Student
Department of Microbiology and Immunology
Temple University
United States