Isolating Cells from Atypical Samples
Preparing a Single Cell Suspension:
When working with solid tissues, it is essential to obtain a single cell suspension, and to ensure that the processing method does not adversely disrupt the cell surface epitopes. The Worthington Tissue Dissociation Guide, as well as published protocols in the literature, can be valuable resources for preparing your single cell suspension.
Once the cells are in suspension, it is important to add DNase I (Catalog #07900) to minimize cell clumping, as typically seen when working with previously frozen samples or following enzymatic tissue dissociation. For further details, please refer to the Technical Tip: DNase I treatment for clumpy cell suspensions.
Filtering the cell suspension, i.e. through a 40 µm Cell Strainer (Catalog #27305), is often necessary to remove cell clumps and debris that can interfere with the efficiency of the isolation.
If necessary, undesired cell populations can be removed prior to starting the cell separation procedure. Centrifugation over a density gradient media, such as Lymphoprep™ (Catalog #07801/07851), may be appropriate for removing unwanted granulocytes and red blood cells (RBCs). RBC lysis with Ammonium Chloride Solution (Catalog #07800/07850) may be useful in some cases as well.
Positive or Negative Selection:
Whether you choose negative selection (to isolate untouched cells) or positive selection (where isolated cells are labeled), will often depend on the downstream application or assay for your cells of interest.
Positive selection is suggested as the preferred isolation approach when dealing with atypical samples. Provided that an extracellular marker is available, STEMCELL Technologies offers a number of options for positive selection:
- Using one of our off-the-shelf positive selection kits, if one is available, and adapting it for use with your sample.
- Contact email@example.com to inquire if a custom manufactured positive selection kit would be more appropriate for your application.
- Using our EasySep™ Human Do-It-Yourself Selection Kit (Catalog #18099) for human samples, or our EasySep™ Do-It-Yourself Selection Kit (Catalog #18098) for samples from other species, if a mouse IgG1 antibody against the marker of interest is available.
- Using one of our EasySep™ Indirect Positive Selection Kits, if an antibody against the marker of interest is available conjugated to Biotin, FITC, PE or APC. Please refer to the Technical Tip: Isolate Virtually Any Cell Type Using EasySep™ Indirect Selection Kits for more information and optimization tips.
If untouched cells are required, then it is essential to assess the frequency and composition of the various cell populations present in your starting sample by flow cytometry, to ensure optimal enrichment. It is important to determine the frequency your desired cells, and the unwanted cell types that need to be removed. Common cell types to look for include T cells, B cells, NK cells, monocytes, granulocytes, dendritic cells and epithelial cells. With this information, the options for negative selection include:
- Using one of our off-the-shelf enrichment kits designed for samples with a matching cellular composition to your starting sample.
- Contact firstname.lastname@example.org and we can help you design a custom enrichment kit that suits your specific needs.
- If you are working with mouse samples, you might also consider obtaining your own biotinylated antibodies, and using these in conjunction with the EasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit (Catalog #19860). Please note that this kit is not suitable for use with samples treated with ammonium chloride for RBC lysis.
For further assistance and information, or to find out if a custom isolation product might be an option for your specific aim, please contact email@example.com.
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Isolating Cells from Atypical Samples