Positive Selection vs Negative Selection for Cell Isolation

In a previous post, I covered the current options for isolating pure cell populations. One immediate question you will have to ask yourself is whether you would prefer positive selection or negative selection (depletion) for the isolation of your cell type of interest.

Positive selection involves the isolation of a target cell population by using an antibody that specifically binds that population. As an example, a positive selection kit for T cells would use an antibody specific for the CD3 molecule on T cells. Negative selection, however, involves the depletion of all cell types except your cell type of interest. With our T cell isolation example, our negative selection kit would likely involve antibodies specific for B cells (CD19), monocytes (CD14), NK cells (CD56), and so on. With the depletion of these cell types we would only be left with our cells of interest, in this case T cells (CD3).

The Advantages of Positive Selection

Positive selection and negative selection each havepositiveSelection their advantages. Positive selection offers greater purity due to the specificity of the reaction. You know in our example that positive selection of T cells will only yield a high purity of T cells due to the binding of selection antibodies to CD3 molecules. Negative selection, however, is inherently leakier since it is impossible to design a perfect depletion cocktail to target all cells that do not carry CD3 molecules. It is important to point out though that all of the popular cell isolation companies have made quite excellent kits that yield good purity levels when done properly. The difference in purity between positive selection and negative selection is roughly 99% to 95% pure, both of which are more than serviceable.

Another advantage of positive selection is that it offers the ability for a follow-up selection, or sequential isolations. Since negative selection works by binding all cells except the target cells with bead-bound antibodies, there is no way to do further isolations with the negative population. However, the negative flow through population from positive isolation will not have bead-bound antibodies and therefore is available for either another positive selection or a negative selection of your choice.

The Advantages of Negative Selection

The disadvantage of positive selection of course isnegativeSelection that your isolated cells will carry bead-bound antibodies. Not surprisingly, the kit manufacturers will tell you that this is not a concern, but it is something you need to keep and mind and use at your discretion. While neither the antibodies nor the beads should activate your isolated cells, it may in some way affect your downstream experiments. If you feel this could be an issue and you would prefer ‘untouched’ cells, then negative selection may be the right choice for you. First, however, be sure the negative selection kit actually depletes all necessary cells in order to achieve a pure target population. Often these kits are designed for common target tissues, such as peripheral bloods, lymph nodes, and spleens. Unfortunately negative selection kits may not work well for other target tissues. For example, my own work involves isolation of T cells from tumor samples. Since stock negative selection kits do not contain depletion antibodies for tumor cells, negative selection is not an option for our assays, and as a result we are forced to use positive selection.

It is important to choose an optimal cell isolation strategy specific to your assay, your target cells, and your tissue source. In my next post I will offer some tips for sorting through the various kits and technologies many companies offer for cell isolation.

Current Options for Isolating Pure Cell Populations

Antibody based isolation kits for isolating immune cell populations have become a standard protocol in the toolbox of every immunologist over the last two decades. In fact, many new scientists are shocked to learn that lymphocytes used to be isolated from PBMCs and other tissue sources by filtering through nylon wool. How archaic! Here I will describe the various options cell isolation technologies available to biologists today.

FACS: Fluorescence Activated Cell Sorting

FACS is the most sophisticated way of isolating various cells of interest from your tissue source. You have the ability to incorporate up to 10 or so different fluorescent antibodies into your stain, which allows you to sort on cells of interest with exquisite precision and specificity. Another powerful tool is the ability of many FACS machines to do four-way sorts or even single-cell sorts.

However, sorting can be relatively time consuming, depending on your sample size and the percentage of cells of interest. Use of FACS machines are also fairly expensive, whether it be your laboratory’s investment in acquiring its own machine and committing to its maintenance or the hourly rates your institution’s core will charge you (averaging around $100 per hour in my experience).

Magnetic Antibody Based Cell Isolation

Cell separation reagents are available from the three main players in the cell isolation kit world: Stem Cell Technologies, Miltenyi Biotec MACS Technology, and Life Technologies Dynabeads. Though the technology varies slightly from company to company, they basically boil down to the same principles. Usually an antibody cocktail will bind either your cell of interest (positive selection) or your cells of non-interest (negative selection). After a short incubation the addition of magnetic nanoparticle beads to your cell mixture then binds the antibodies from the previous incubation. After another short incubation, cells can then be placed into the magnet purchased from the company. After a few minutes, the antibody bound cells will be drawn towards the magnet and the unbound cells can be collected. Bound cells can then be washed out and collected separately. This technology allows rapid and easy isolation of cell populations from bulk populations.

However, magnetic antibody based cell isolation involves some upfront investment in the purchasing of magnets (approaching $1000) and antibody kits (ranging from $300-$700). Because of this it is important to fully research which companies’ technology is right for you. I also highly recommend sampling the technology on some extra PBMCs you have if at all possible and finding an experienced colleague that can advise when you have questions.

RosetteSep Whole Blood Based Cell Isolation

RosetteSep kits from Stem Cell Technologies allow researchers to quickly isolate cells of interest directly from whole blood and without the investment in magnets. Furthermore it combines the Ficoll gradient isolation step with the isolation of specific target cells, making for an efficient and economical protocol. Instead of using magnetic nanoparticles, RosetteSep uses antibodies that conjugate directly to the RBCs in whole blood. When the blood is Ficolled the RBCs go to the bottom layer along with all the cells that you have targeted with antibody. Your top layer is left with untouched cells of your interest! Of course this protocol only works from whole blood, so it will not work on PBMCs or cells from other tissue sources.

Keep in mind that both FACS and antibody based cell isolation require starting with a single cell suspension of cells. It is important to think about whether you want touched or untouched cells (positive or negative selection) for your downstream assays. I also highly recommend doing purity checks (see figure below) by flow cytometry as often as you can, especially when first adapting any isolation technology to your lab.

Stemm Cell CD14 iso resized 600

 These powerful techniques allow for biologists to isolate a host of cells, including T cells, B cells,  Monocytes, Stem Cells, and many more. In an upcoming post I will go into even further detail and how to choose the right technology for you, including some of the tips and tricks I have learned in my own experience

Further Reading:

Stem Cell Technologies: http://www.stemcell.com/en/Products/Product-Type/Cell-isolation-products.aspx

Life Technologies Dynabeads: http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Dynal/Dynabeads-Types-and-Uses.html

Miltenyi Biotec MACS Technology: https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation.aspx

RosetteSep: http://www.stemcell.com/en/Products/Popular-Product-Lines/RosetteSep.aspx

Maturing and Assaying Monocyte-Derived Dendritic Cells

Generating dendritic cells (DCs) from PBMC CD14+ monocytes allows researchers to do a host of immunological assays. A common example of this is to examine the reactivity of a T cell mixture to a certain antigen of interest. However, prior to doing such antigen presentation assays, DCs must be properly matured in order to fully elicit a T cell response.

After generating your monocyte derived DCs (mDCs) from PBMCs, as I described in my previous post, you will have several choices on how to mature them. Two of the most common choices are to either use LPS or a monocyte maturation cocktail (MMC). LPS binds TLR4, which results in a host of downstream inflammatory genes being upregulated. Addition of IFNγ can polarize the DCs to a Th1 phenotype, while the addition of TNFα can polarize the DCs somewhat towards a Th2 phenotype. MMC, however, usually involves the addition of several molecules including TNFα, IL-6, IL-1β, and PGE2. The overall effect of this pool of molecules is to elicit a mixed Th1 and Th2 response by the DCs. Thus, the maturation method of choice is a critical choice for the researcher and may vary depending on the downstream functional assay.

Interrogating your DCs by flow cytometry is a good idea so you can be sure you have attained the cell phenotype you desire. mDCs will commonly express CD11c and CD1c and should be CD123-. Furthermore, upregulation of costimulatory molecules CD80 and CD86 and the immunoregulatory molecule CD83 and downregulation of CD14 are hallmarks of DC maturation. HLA molecules are also significantly upregulated. Remember, these molecules are not just cell markers, but have important functional relevance. The upregulation of costimulatory molecules is critical for the activation of T cells and the upregulation of surface HLA molecules is a reflection is the enhance antigen presentation capability of a mature DC.Dendritic Cells Dot Plot with CD1c and CD11c Expression

Running your DCs on the flow cytometer will require a few special tweaks on your normal cytometer settings. The first thing you will notice is that the DCs are rather massive and irregular shaped cells. You will therefore likely need to significantly decrease both your forward scatter and side scatter to locate them on your dot plot. Secondly you will want to significantly decrease the voltages for all the channels detecting fluorchromes on your DC activation markers. These activation markers are expressed at such a high level on the DCs, that they are incredibly bright. A third issue is the high level of auto-fluorescence on DCs. It is always a good idea to have some extra DCs you can run while setting up your voltages to make sure your CD marker fluorochromes are all on scale.  Be sure to use the activated sample of DCs for this! Once you have verified your settings will work, you can then proceed to normal compensation set up.

Once your cytometer settings are established your cells are ready to assay. It is a good idea to have a sample of DCs that you did not stimulate as a control to compare your matured DCs to. In my experience the best way to compare markers, such as CD83, CD86, HLA-ABC, and HLA-DR, is by using histogram overlays. Their upregulation can often be a slight shift in fluorescent intensity, which you can readout by graphing Median Fluorescent Intensity (MFI). Of course be sure that you have titered your antibodies appropriately and use isotype controls when you can. Also keep in mind that comparing MFI readouts between different assay days, different stains, and different experiments is virtually impossible. Try to group your assays whenever possible, but if not, fold change in MFI is a useful, though not ideal, calculation for comparing these sorts of data.


Differentiation of Peripheral Blood Monocytes into Dendritic Cells. David W. O’Neill, Nina Bhardwaj. Current Protocols in Immunology. July, 2005.

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. Bender A, Sapp M, Schuler G, Steinman RM, Bhardwaj N.  J Immunol Methods. 1996 Sep 27;196(2):121-35.

Monocyte-derived DC maturation strategies and related pathways: a transcriptional view. Luciano Castiello, Marianna Sabatino,   Ping Jin, Carol Clayberger, Francesco M. Marincola, Alan M. Krensky, David F. Stroncek. Cancer Immunol Immunother. 2011 April; 60(4): 457–466.

Taking dendritic cells into medicine. Steinman RM, Banchereau J. Nature. 2007;449:419–426.

Current approaches in dendritic cell generation and future implications for cancer immunotherapy.  Tuyaerts S, Aerts JL, Corthals J, et al. Cancer Immunol Immunother. 2007;56:1513–1537.

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines.  Dohnal AM, Graffi S, Witt V, et al. J Cell Mol Med. 2009;13:125–135. 

describe the imageColt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.

Generation of Dendritic Cells from Peripheral Monocytes

describe the imagePBMCs are not just a source of many different circulating immune cell types, but also a source of potential cells that one can generate in vitro. One excellent and long-standing example of this is the generation of dendritic cells (DCs) from monocytes.  Monocyte derived DCs (mDCs) are an excellent tool for researchers to do immunological assays requiring a source of professional antigen presenting cells (APCs). While circulating B cells are capable of antigen presentation and T cell activation, they do not offer the robust response that DCs do. The generation of mDCs is a relatively simple protocol that anyone can do with just a source of PBMCs, a few important cytokines, and, of course, some media and incubator space. After this protocol, you will have obtained immature mDCs that can then be matured for use as APCs in your assay.

The first step in generating mDCs is to decide how you would like to isolate the monocyte population from your PBMCs, which serve as your precursor cells for DCs. The easiest and cheapest way is to simply plate your PBMCs on a cell culture dish and let the inherent qualities of monocytes go to work. Monocytes are unique amongst other PBMC cells in their tendency to stick to plastic. An incubation period between 1-24 hours will allow your monocytes to adhere to the dish and let you gently wash off any other PBMCs. The alternative to the adherence method for isolating monocytes is to use a magnetic antibody based technology of your choice. Several companies, such as Miltenyi Biotec, Life Technologies, and Stem Cell Technologies, offer excellent kits for this. While the adherence method is cheaper, antibody based kits give you higher monocyte recovery and purity, which may or may not matter depending on your downstream assays.

Once you have your monocytes isolated from your PBMCs, you can begin the 7 day culture to generate mDCs. Monocytes can be plated in a standard cell culture media along with two important cytokines, GM-CSF and IL-4 (50ng/mL and 100ng/mL). GM-CSF will push the monocytes down a DC differentiation pathway. IL-4 will inhibit the monocytes from differentiating into macrophages, thereby insuring they become DCs. Continue the culture for 6-8 days and be sure to refresh your cytokines every other day.

As the monocytes differentiate over the culture period, note their progress by examining them with your tissue culture room microscope. The cells should appear as fairly round and are generally 2-3 times the size of lymphocytes. It is important to note that the mDCs will not appear like the elongated cartoon DCs with long extensions you see in text books. Those DC characteristics are generally only found in tissues and not in vitro.  While you may see some cells that resemble this, those are more likely to be somewhat of a natural stromal layer, made up of cells including macrophages, that the monocyte culture develops to support cell growth. In fact, the immature mDCs will have very few if any, cytoplasmic protrusions.

DC2 resized 600Once the culture period has finished, between 6-8 days, the mDCs can be collected. The exact day is not critical, as long as you remain consistent in the day you pick for your following experiments. To collect the mDCs, gently wash the culture dishes with several streams of media by pipetting up and down. The mDCs, which are currently immature, will be somewhat floating and only loosely adherent. Because of their loose adherence, they require several rounds of gentle pipetting, but do not require cell scraping, EDTA, or trypsin treatment. Note that the culture dishes will still contain some adherent cells. Do not worry about these cells, since these are not the loosely adherent DCs we are interested in.

After completion of these steps, you should have a nice population of immature mDCs, which express CD11c, CD1c, and are CD123-. In my next post, I will cover some tips and tricks for analyzing these cells by flow cytometry. Importantly, I will also cover ways to mature the immature mDCs for use as APCs.

Colt EgelstonColt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.