RosetteSep® Human CD4+ T Cell Enrichment Cocktail From StemCell Technologies

RosetteSep® Human CD4+ T Cell Enrichment Cocktail is the best tool in the market to enrich for CD4+ T cells from human blood. The relatively simple principle behind the success of this tool is to crosslink by immunorosette formation and separate unwanted cells from human whole blood by density gradient centrifugation in order to give a final yield of a highly pure fraction of CD4+ T cells. The RosetteSep® antibody cocktail covers a wide range of cell surface antigens present on cell populations that normally contaminate purified lymphocyte cell preparations. The highlight of the technology is the significant yield of virgin population of CD4+ T cells from large volumes of human blood in a very short time.

We have been using this cocktail routinely for isolation of T cells from blood. From 50 ml of human blood, we are able to purify 20-30 x 10⁶, 92-96% pure CD4+ T cells in 90 minutes from start to finish, consistently. We add 400ul of Rosette Sep cocktail to 10 ml of blood and mix it well. After a 20 minute incubation at room temperature, 10 ml of PBS + 2% FBS is added and mixed gently. The diluted blood cocktail mixture is then layered on density medium and centrifuged for 20 min at 1200 x g. The enriched cells are then collected from the interface.

Our lab focus is to evaluate the potential of certain cell populations to modulate the proliferation of CD4+ T cells. Cell sources for our studies are patient samples which are hard to come by and very precious. Hence this easy to use, efficient, consistent and most reliable research tool has been very beneficial for our experiments. Most of the available lymphocyte purification systems, including magnet based and antibody based methods, do give pure T cell fractions. However, in our experience, these methods also compromise the viability of the purified T cell fractions. The RosetteSep cocktail offers the advantage of purifying untouched and viable cells consistently. Hence we strongly recommend this cocktail for research purposes. We have not observed lot to lot variability for this product which is a significant plus for researchers.

However, this tool is relatively expensive. But the call one has to make is whether to compromise quality of your data for expense. This is particularly important for studies involving human samples. In our studies, we have been able to modify some of the requirements proposed by StemCell Technologies. We have been able to replace Ficoll or DM-L with a relatively inexpensive lymphocyte separation medium (Lymphocyte separation medium - Density 1.077-1.080 g/ml at 20°C, from Mediatech, Inc.) and still recover a highly pure fraction of CD4+ T cells. The protocols have also been improvised to use less (We use 2 ml of cocktail for labeling 50 ml of blood.) of the cocktail than the product recommendations to give the same yield of enriched T cells.

Although a relatively simple PBMC isolation protocol could also recover lymphocytes from blood, the isolates are significantly contaminated with myeloid cells which would significantly confound the data by their effects on T cell responses in in vitro culture experiments. However, one has to be careful in evaluating whether a pure fraction of T cells is very important for the experiments that you have designed to specifically address the questions that you are asking in your study.