Complement Mediated Cell Depletion-Protocol & Troubleshooting
Immune cell populations have unique functions. In order to study them, immune cells first need to be purified. Complement depletion is a common purification strategy. This method is rapid, low cost and high yield.
Purification of cell populations is a common procedure in our daily experiments. Here, Creative Biolabs describes a method and basic protocol for the rapid and efficient depletion of cell populations using antigen-specific antibodies and complement depletion.
Solutions and Reagents
| Stages | Solutions and Reagents |
| Cell Preparation | Culture media |
| Cell Depletion | Antigen-specific antibodies, rabbit complement, culture medium |
Complement Mediated Cell Depletion Procedure
The complement system consists of a large number of plasma proteins. When the complement system is activated by antibodies, a protein hydrolysis cascade reaction is initiated. The complement cascade can cleave any cell population in an antigen-specific manner. Thus, we can achieve cells depletion by adding antibodies and complement to cell populations.
1. Cell Preparation
Collect cell samples. Then adjust the number of starting cell populations in the culture medium.
2. Antibody Selection
First, select antibodies that are effective in activating complement and test their activity. Then, add each antibody to the cell suspension at an appropriate predetermined concentration. Finally, mix the cells well by inverting them back and forth.
3. Adding Complement
First calculate the amount of complement required. Thaw the complement and sterilize it. Then add the complement directly to the tube containing the cells and antibodies.
4. Cell Purity Determination
Adjust the volume of cells in the tube and incubate the cells. At the end of the incubation, centrifuge the cells and discard the supernatant. Then collect the cell precipitate by multiple washes. Finally, check the cell purity by comparing the cell population of interest before and after depletion.
Troubleshooting
Poor cell purity
-
Antibody causes. The most critical issue in using complement removal cells is the need for antibodies that are complement-activating with the desired antigen specificity. You must test in a pre-experiment to determine the effectiveness of the different species and types of antibodies you choose.
On the other hand, you may have already identified antibodies. However, its inefficiency leads to poor purification. We suggest that you can perform two complement depletions without affecting the viability of other cells. As well, you can pre-titrate the antibody already to obtain the optimal antibody concentration. - Complement causes. First, check the quality of the complement reagent you purchased to make sure it is highly active. It is worth noting that you will need to thaw it or need to sterilize it before using it to add it to the cell suspension. Again, we recommend titrating the complement to determine the optimal working concentration. Too high a concentration can affect cell viability.
- Incubation time causes. Incubation time is equally important. We recommend an incubation time of no more than 60 minutes.
Purification targeting specific cell populations is a common experimental need. Many strategies can be applied. Complement depletion can be performed quickly in any laboratory and does not require expensive equipment or reagents. If you would like to try this fast and effective protocol, you can order our products and follow the protocol.
Reference
- Dittel B N. Depletion of specific cell populations by complement depletion. J Vis Exp, 2010;(36):1487.
For research use only. Not intended for any clinical use.
Send Inquiry
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
