How to Calculate Cell Recovery & Measure Purity After Cell Isolation
Setting aside a small portion of your starting sample allows you to assess the frequency of desired cells in the sample prior to isolation
Accurately assessing your starting sample, along with calculating cell recovery and measuring cell purity, are essential steps for evaluating the success of any cell isolation workflow. Whether you’re working with peripheral blood mononuclear cells (PBMCs), cord blood, bone marrow, tissue-derived single-cell suspensions, or any other cell source, this guide provides tips to help you determine cell recovery and purity, supporting consistent and reliable results.
Why Assess Your Starting Sample?
Setting aside a small portion of your starting sample immediately before cell separation is very important. This will allow you to assess the frequency of desired cells (i.e. purity) prior to isolation, and supports accurate calculation of cell recovery following isolation.
Tip: Always keep a small aliquot of your starting sample to establish a baseline and compare pre- and post-isolation results.
Key Measurements to Assess in Your Starting Sample
To accurately assess your starting sample and support downstream calculations, it is important to evaluate the following parameters:
- Total nucleated cells (TNCs): This is the total number of cells in your sample containing a nucleus. TNC counts define the starting input and are used alongside post-isolation counts to calculate cell recovery.
- Cell viability: This is the percentage of live cells in your sample. Measuring viability before isolation is essential for determining the quality of your starting sample and assessing how much your isolation method affects cell viability.
- Frequency (%) of target cells: This is the proportion of your desired cell population within the sample prior to isolation. This measurement supports interpretation of cell purity and calculation of cell recovery after isolation.
Expected cell frequencies in various tissues and cell sources can be estimated based on historical and published data. However, the actual frequency of your cells of interest can vary from donor to donor and depends on whether the sample is from a normal, diseased, or genetically modified source. To support your planning, download the following free wallcharts on cell type frequencies:
What Is Cell Recovery?
Cell recovery measures how efficiently your desired cells were retained following cell isolation, and it is a key metric for evaluating workflow performance and optimizing your protocol. While often used alongside related terms, it is important to distinguish between them:
- Cell recovery: The percentage of target cells retained after isolation (efficiency)
- Cell yield: The total number of target cells obtained after isolation (output)
- Cell throughput: The total number of cells or samples that can be processed or obtained; particularly relevant when scaling workflows or working with large sample volumes
How to Calculate Cell Recovery
Calculating cell recovery requires four key values, and accurate determination of each is critical:
- Total number of cells in the starting sample
- Percentage of desired cells in the starting sample (i.e. starting cell purity)
- Total number of cells in the enriched fraction
- Percentage of desired cells in the enriched fraction (i.e. final cell purity)
Note: Cell Yield = Total Cells in Enriched Fraction × Final Cell Purity (%)
Tip: For convenience and to reduce variability, analyze your saved starting sample aliquot alongside your enriched fraction at the end of your experiment.
How to Count Cells Accurately
To determine the total number of cells in both your starting sample and enriched fraction:
- Always use the TNC count for recovery calculations
- Assess cell viability for accurate results
- If using automated counters, ensure red blood cells (RBCs) are excluded and only white blood cells are counted
How to Count Viable vs. Total Nucleated Cells
- Trypan Blue (Catalog #07050) is commonly used to assess viability in mammalian cells. It enters cells with compromised membranes (dead cells), staining them blue, while live cells remain unstained.
- Using Trypan Blue for samples containing red blood cells (RBCs) makes it difficult to distinguish between nucleated cells and RBCs. In these cases, use 3% Acetic Acid with Methylene Blue (Catalog #07060). This solution lyses cell membranes, leaving nuclei lightly stained, allowing you to count white blood cells specifically (as mature RBCs lack nuclei).
Tip: We recommend using 3% Acetic Acid with Methylene Blue to accurately count cells in samples containing RBCs.
For more details on cell counting, please see our protocol and associated video:
How to Count Cells with a Hemocytometer
Learn how to perform total nucleated cell counts using 3% Acetic Acid with Methylene Blue, and how to perform viable cell counts by Trypan Blue dye exclusion. Additionally, explore cell counting resources and templates to help streamline your assays!
What Is Cell Purity?
Cell purity refers to the frequency (%) of your desired cells within a sample. It is a key metric for evaluating the effectiveness of your isolation process and the quality of your enriched fraction.
How to Measure Cell Purity
Cell purity is measured using flow cytometry by labeling your target cells with fluorescent antibodies and identifying them through a gating strategy. Gating helps isolate your population of interest while excluding debris, dead cells, and unwanted cell types, enabling accurate calculation of the percentage of target cells in your sample. This measurement is essential for confirming cell isolation performance and ensuring your sample is suitable for downstream applications.
Considerations for Flow Cytometry Gating
Learn key considerations for flow cytometry gating to help ensure accurate identification and analysis of your target cell populations.
Tip: How to Get Accurate Cell Purity Results
- Always gate on viable cells to ensure accurate purity measurements
- Process starting and enriched samples in parallel when possible
- When using ·¡²¹²õ²â³§±ð±èâ„¢ for cell isolation, follow the kit-specific recommendations
Refer to the Product Information Sheet, “Notes and Tips,†of each specific ·¡²¹²õ²â³§±ð±èâ„¢ kit for recommended methods. Certain positive selection kits may introduce antibody blocking effects; therefore, following specific instructions is critical for accurate purity assessment, for example:
- Using appropriate antibody clones
- Adding the staining antibody at the same time as the cell separation cocktail
- Following kit-specific staining instructions
Simplifying Your Cell Isolation Workflow
Learn how ·¡²¹²õ²â³§±ð±èâ„¢ enables efficient, column-free cell isolation using immunomagnetic separation to obtain highly purified cells in minutes.
Additional Resources
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