Complement depletion is a fast, effective, and inexpensive method for purifying immune cell populations. Complement depletion begins when monoclonal IGM or IgG antibodies are added to a cell population, which target a specific cell type. In this example, we will target T-cells for depletion.
The complement system is then added initiating a proteolytic cascade, which results in T-cell death. The cell population is then centrifuge washed and filtered to eliminate dead cells and debris. This complement depletion method typically results in the elimination of 95 to 100%of the targeted cell population.
Hi, I'm Bonnie Dittle. I'm an investigator at the Blood Research Institute of Blood Center of Wisconsin located in Milwaukee. Today I'm going to show you the technique complement depletion, a fast and inexpensive method for the depletion of specific cell populations.
We use this technique in my laboratory to highly enrich immune cell populations that then we use for a number of bioassays, including those involving cell activation and protein expression analysis. So let's get started. Before beginning the experiment, prepare 0.5 or one milliliter aliquots of rabbit complement and store in either a minus 20 or minus 80 degree freezer.
Be sure to titrate the complement to an optimal dilution between one to 10 and one to 20. An excellent commercial source for complement that we routinely use is from Pereze biologicals for this procedure, mouse and rap monoclonal antibodies are generally effective at complement activation, but not all antibodies will be suitable. IgM antibodies are generally the most efficient for complement induced cell lysis.
IgG antibodies are the second most efficient and are typically used because most monoclonal antibodies are IgG titrate each antibody for optimal use in a cell density of 10 to the seventh cells per milliliter, which is determined empirically. Most antibodies are used in the range of 0.1 to 10 micrograms per milliliter. For the depletion of splenic T-cells, we use a monoclonal antibody specific for TH one.
To begin complement depletion dilute the cells in cell culture medium containing 5%heat inactivated fetal calf serum so that the starting cell population is two times 10 to the seventh cells per milliliter. For example, five times 10 to the seventh cells should be diluted in 2.5 milliliters of medium. This volume of cells is one half of the final volume, and hence twice the concentration of cells used during depletion, which will be one times 10 to the seven cells per milliliter.
At this time, save up to one times 10 to the six cells so that the depletion can be quantified at the end of the experiment. In addition, thaw the required amount of complement at room temperature. We have found that a dilution of one to 15 is appropriate when using pereze biologicals rabbit complement for five times 10 to the seven cells, 1.5 milliliter frozen Eloqua is sufficient.
Now add the appropriate amount of antibody to the cell suspension and mix. In order to sterilize the complement, use a low protein binding syringe tip filter. Fill the syringe with one to two milliliters of medium.
Before adding the complement for five times 10 to the seven cells, we will need 340 milliliters of complement for a one to 15 dilution. Then filter the complement directly into the tube containing the cells and antibody. Now adjust the final volume in the tube so that the cells are at a concentration of one times 10 to the seventh cells per milliliter.
Next, allow the cells to incubate at 37 degrees Celsius in a water bath for one hour. Remember to mix the cells every 15 minutes by gentle inversion. After the incubation, centrifuge the cells at a speed that will not affect viability.
Then back in the hood, discard the supernatant. Next, wash the cells twice with 10 milliliters of medium. Notice how the cell debris collects with some sticking to the sides of the polystyrene tubes.
After the last wash, filter the cells through a 70 micron sterile nylon cell drainer. Finally, check the cell purity. By comparing the cell population of interest before and after depletion flow.
Cytometry and immunohistochemistry are preferred methods used to determine the cell purity prior to experimentation. When using a monoclonal antibody that's highly efficient at activating complement, the first round of complement depletion will deplete the cells by more than 95%As an example, using flow cytometry to measure the percentage of alpha beta T cells in a mouse ple cyte cell preparation, we found they contained 32.2%alpha beta T-cell following complement depletion with anti five one, which is expressed by all T cells, but not by other leukocytes. The TCR beta population was reduced by 95%to a final concentration of 1.56%sent.
I've just shown you a fast and effective method of depleting specific cell populations using antigen specific antibodies in complement depletion. When doing this procedure, remember to test each antibody for effectiveness and to titrate your complement in a pilot experiment before you begin. That's it.
Thank you for watching and good luck with your experiments.
