The mouse T cell enrichment kit can be used for the purification of T cells from mouse splenocyte samples from which erythrocytes have been lysed with a red blood cell lysis buffer (NH4CL2). Provided with the kit are the columns as well as the column wash buffer. The kit works via negative selection. The glass beads in the column are coated with surface anti-immunoglobulin antibodies (anti-Ig) and immunoglobulins (Ig). B cells bind to the anti-Ig and monocytes bind to the Ig via Fc receptor interactions. Cell populations running through the column are enriched for T cells and can be directly used for tissue culture or other experiments. A portion of the starting cell population should be kept to monitor the enrichment process, i.e. analysis of the starting and the eluted cell populations by flow cytometry with anti-CD3 (T cell) and anti-CD19 (B cell) antibodies..
Each column can be used for up to 100 x 106 cells. The cells which need to be enriched for T cells should be re-suspended in 1 mL of the column wash buffer (1x). The column is placed in a column rack or ring stand. First, the top cap of the column is removed to avoid drawing air into the bottom of the column. The top is a snap cap which might be difficult at first to take off. Next, the bottom cap is removed. Then, the fluid within the column is allowed to drip into a waste receptacle. After washing the column beads with 6 mL of 1x column wash buffer, the column is ready to be loaded with cells. A fresh, sterile 15 mL centrifuge tube is placed under the column and the 1 mL cell suspension is applied onto the column. The cells now suspended within the column are incubated at room temperature for 10 minutes. After this incubation time, the cells are eluted from the column in four 2ml aliquots of 1x column wash buffer. The collected fractions are centrifuged at 250 x g for 5 minutes. Eluted T cell enriched cells are re-suspended in the respective culture medium, counted and ready for use.
The whole procedure takes about 1h time and several columns can be easily run in parallel which will increase the time used for the T cell enrichment. The flow rate of the column might differ between columns run in parallel as this depends on how many cell clumps or cell debris are present in the actual samples. However, since the white filter at the top of the column bed will stop buffer flow, it is very easy to use several columns in parallel since they can not run dry. At least not so quick.
Mouse T Cell Enrichment Columns from R&D Systems
As the column flow rate is dependent on the quality of the cell sample the procedure might take longer than expected. Also the eluted cell numbers might be quite low.
The Bottom Line
Straight forward column procedure.