Name: Depletion of Specific Cell Populations by Complement Depletion (Video) | JoVE Journal
Uploaded: 2010-02-05T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Learn complement depletion for immune cell purification using monoclonal antibodies and rabbit complement. This method achieves 95-100% elimination of targeted T-cells for downstream bioassays.

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I would like to thank Sreemanti Basu for providing the data for Figure 1. This work was supported in part by NIH grant AI069358 and the BloodCenter Research Foundation.

A. Rabbit Complement
<ol>
<li>Stock rabbit complement should be thawed at room temperature, and then aliquoted into 0.5 and/or 1 ml volumes and frozen at -20-80 °C for long-term storage.</li>
<li>Each lot of complement should be titrated to determine the optimal working concentration. Typically, a dilution of 1:10-1:20 is optimal.</li>
<li>Rabbit complement is not always presterilized prior to sale, depending on the final use of the cells, the complement should be filtered sterilized when aliquoted and frozen or prior to addition to the cells.</li>
</ol>
B. Antibody Selection
<ol>
<li>Not all antibodies are effective at complement activation, thus the antibodies must first be tested for activity.</li>
<li>Both mouse and rat mAb are effective at complement activation.</li>
<li>IgM antibodies are generally the most efficient for use in complement-induced cell lysis followed by IgG. The IgG isotypes best for complement activation vary between species.</li>
<li>Each antibody should be titrated for optimal use at a cell density of 107 cells/ml.</li>
</ol>
C. Basic Protocol
<ol>
<li>Adjust the starting cell population to 5 x 107 cells/ml in medium containing heat inactivated FCS. The final volume of the cells will be twice the starting volume. Remember to save some cells so that the depletion can be quantified.</li>
<li>Each antibody should be added to the cell suspension at the proper predetermined concentration or dilution. Mix the cells well by inversion.</li>
<li>Calculate the amount of complement needed. Thaw the complement stock at room temperature. If the complement is not sterile, sterilize it using a low protein binding syringe tip filter. To the syringe, add several ml of medium prior to the addition of the complement and filter directly into the tube containing the cells and antibody.</li>
<li>Adjust the final volume in the tube so that the cells are at 1 x 107 cells/ml.</li>
<li>Incubate the cells at 37 °C for one hour, mixing every 15 minutes.</li>
<li>Centrifuge the cells and discard the supernatant.</li>
<li>Wash with medium twice. Cell debris will stick to the side of polystyrene tubes. Dead cells and debris can be eliminated using a cell strainer or by density gradient centrifugation.</li>
<li>Check the cell purity by comparing the cell population of interest before and after depletion. Flow cytometry and immunohistochemistry are two good methods of determining cell purity.</li>
</ol>
Representative Results
When using a mAb that is highly efficient at activating complement, cell depletion after one round is greater then 95%. This is shown in Figure 1 where we started with mouse splenocytes composed of 32.2% αβ T cells as detected by expression of TCRβ (eBioscience, San Diego, CA) by flow cytometry. We depleted T cells using a mAb specific for Thy1, a protein expressed by all T cells, but not other leukocytes. The antibody we used was Y-19 a rat IgG2c2. Following the procedure described, the TCRβ population was reduced to 1.56%, a greater than 95% reduction.
<img src="/files/ftp_upload/1487/1487fig1.jpg" alt="Flow cytometry charts of TCRβ expression before and after cell depletion, displaying cell count changes." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Depletion of thy1+ splenic T cells by complement activation. Total mouse splenocytes were obtained by homogenization of the spleen and lysis of RBC. Anti-thy1 and baby rabbit complement at predetermined concentrations were added to a single cell suspension of splenocytes such that the final cell concentration was 1 x 107 cells/ml. After incubation for 1 hour at 37 °C, the cells were washed twice and evaluated by trypan blue exclusion for cell viability. The efficiency of depletion was assessed by flow cytometry comparing the percentage of TCRβ+ cells before depletion (left panel) and after depletion (right panel). The horizontal bar indicates positive staining and the number above the bar is the percent positive.

<ol><li>Janeway, C., Travers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Immune+System+in+Health+and+Disease%5BTitle%5D)+AND+%22Immunobiology%22%5BJournal%5D)">The Immune System in Health and Disease.</a> Immunobiology. , Current Biology Limited; Garland Pub. Inc. London; San Francisco; New York. (1994).</li>
<li>Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+activities+of+antibodies+against+brain-associated+T+cell+antigens+II.+Stimulation+of+T+cell-induced+B+cell+proliferation%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation.</a> Eur J Immunol. 12, 30-37 (1982).</li>
</ol>

The purification of cell populations is a common procedure required for the study of their functions. Here we describe a fast and effective method of depleting cell populations using antigen-specific antibodies and complement depletion. The depletion of any cell population can be achieved if a complement-activating antibody is available to a lineage specific marker. Here we demonstrated the depletion of T cells using a mAb specific for Thy1, a protein expressed by essentially all T cell populations. We depleted over 95% of &alpha;&beta; T cells and the remaining splenocytes were increased from 68% to 98%. This level of cell purity is similar to other purification strategies including FACS and magnetic bead separation. Complement depletion is a fast technique that can be performed in 1.5 hours once the starting cell population is obtained. Another advantage of complement depletion is that any laboratory can perform the technique because it does not require expensive equipment and reagents. The only major pieces of equipment required are a water bath and a centrifuge. Rabbit complement can be purchased from a variety of vendors at a reasonable cost (see table of reagents) and many mAb can be purchased from ATTC and grown locally to control costs. In contrast, FACS requires a flow cytometer with cell sorting capacity that will cost in the six figures and expensive fluorescently tagged mAb. Magnetic bead separation is moderately priced, but requires the purchase of a magnet and magnetic beads, which can be costly. In addition, some magnetic bead separation strategies also require the purchase of columns.
The main stumbling block to the use of complement for cell depletion is the availability of complement activating mAb with the antigen specificity required. Although human, mouse and rat antibodies all have the capacity to fix complement, some isotypes are better than others. Although IgM from all three species is highly effective at fixing complement the IgG isotypes vary. Thus every mAb must first be tested in a pilot study to determine its effectiveness. For antibodies that are not highly efficient, two rounds of complement depletion can be performed without affecting the viability of the remaining cell populations. As a cost savings, the antibodies should be titrated for optimal effectiveness. In addition, the antibodies used do not need to be purified. Hybridoma supernatants and ascites fluid work just as effectively, which is particularly convenient for IgM antibodies, which are difficult to purify. As with the antibodies, it is imperative that the complement be titrated in a pilot study. A too high of concentration will result in loss of viability of non-targeted cell populations. Also important is that the incubation time does not exceed 60 minutes.
In our protocol, we co-incubate the antibody and the complement together. However, the cells can be labeled with antibody for 15-30 minutes and washed prior to the addition of complement. When using this strategy, make sure to perform the antibody incubations on ice to prevent the loss of the cell antigen on the cell surface, as occurs with some proteins by patching and capping or receptor internalization. Although highly efficient, the main drawback to complement depletion as compared to other methods is that it cannot be used for positive selection. Thus it is difficult to obtain subpopulations of cells. For example, it would be difficult to purify marginal zone B cells from follicular B cells, which would best be done by FACS. In this situation, complement activation could be used to deplete T cells to reduce the number of cells being sorted. This strategy would reduce the sort time, which would save money and would be less harsh on the cells.
Since the purification of specific cell populations is a common requirement a number of strategies have been developed. The main advantages of the complement depletion strategy are its affordability, technical simplicity and time savings.

<table><tbody><tr><td>3-4 week baby rabbit complement</td><td>Pel-Freez Biologicals</td><td>31061-3</td><td></td></tr><tr><td>Water bath</td><td>Various</td><td>various</td><td>An incubator set at 37 &amp;#x00B0;C can also be used.</td></tr></tbody></table>

depletion of specific cell populations by complement depletion

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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.

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Learn complement depletion for immune cell purification using monoclonal antibodies and rabbit complement. This method achieves 95-100% elimination of targeted T-cells for downstream bioassays.

Depletion of Specific Cell Populations by Complement Depletion

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