Release date:
2025-11-13 View count: 8
When analysing peripheral blood samples by flow cytometry, deciding whether to stain or lyse the cells first is a key experimental decision that significantly impacts the results. The goal is to remove the majority of red blood cells without altering the true state of the white blood cells, while ensuring specific fluorescent labelling. The order in which these steps are carried out can impact cell yield, antigen preservation, background fluorescence, and the overall reliability of the data.
1. Common Protocols for Peripheral Blood Processing
1.1 Bulk Lyse (Lyse-Stain-Wash)
In this approach, anticoagulated whole blood is first treated with a red blood cell lysis buffer, followed by incubation for a specified period. After centrifugation, the supernatant containing haemoglobin and red blood cell debris is discarded, leaving behind the white blood cell pellet. The cells are then resuspended, counted, and subsequently stained with antibodies. After incubated, the cells are washed, resuspended, and detected.
1.2 Tube Lyse (Stain-Lyse-Wash)
For this protocol, the whole blood is first incubated with flow cytometry antibodies, allowing them to bind to cell surface antigens. After staining, red blood cells are lysed using a lysis buffer. The cells are then centrifuged, washed to remove debris and unbound antibodies, and detected.
Although these two protocols differ only in the order of staining and lysis, both are commonly used depending on the specific needs of the experiment. So, how does the order of these steps influence the outcomes?
2. How the Order Affects Experimental Results
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Factor
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Lyse-Stain-Wash
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Stain-Lyse-Wash
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Cell Environment
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Utilises an artificial buffer, which may not fully reflect in vivo conditions.
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More closely mimics in vivo conditions.
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Cell Yield and Integrity
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Lower cell yield; repeated centrifugation and washing can lead to cell loss, especially for rare or fragile cells.
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Higher cell yield with better preservation of cell integrity, as fewer washing steps are involved.
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Antigen Integrity
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Lysis buffer can damage sensitive epitopes, potentially affecting antibody binding.
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Staining before lysis helps protect epitopes from potential degradation.
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Antibody Efficiency
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Direct binding to white blood cells increases antibody utilisation, reducing the need for large volumes.
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Some antibodies may bind non-specifically to red blood cells or plasma proteins, reducing efficiency.
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Operational Efficiency
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Centralised cell counting and concentration adjustment after lysis allows for efficient processing, making it suitable for high-throughput experiments.
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Each sample requires separate staining and lysis, making this approach more labour-intensive.
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Cell Concentration Adjustment
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Lysis allows for flexibility in adjusting cell concentration, which is helpful for concentrating samples or enhancing detection of rare events.
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Limited flexibility for adjusting cell concentration after staining.
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From the comparison, it’s clear that Lyse-Stain-Wash is more efficient for large-scale experiments requiring high throughput, antibody economy, and sample concentration. It’s ideal for multi-tube, multi-colour studies. On the other hand, Stain-Lyse-Wash is preferable when it’s critical to preserve cell integrity, protect sensitive epitopes, and minimise the loss of rare or fragile cells. In essence, the former is more advantageous in terms of "efficiency" and "resource utilisation," while the latter ensures better "data integrity" and "cell preservation."
3. Choosing the Right Protocol for Your Experiment
Each protocol offers distinct benefits, making them more suitable for different experimental needs and applications. The decision should be based on factors such as the specific goals of the experiment, the type and quantity of cells available, cost constraints, and the required level of result accuracy.
1) Sample Volume
Sufficient sample volume: The "Lyse-Stain-Wash" approach is more efficient for handling larger volumes, allowing bulk processing and antibody savings.
Limited sample volume: The "Stain-Lyse-Wash" protocol is better for small samples, as it preserves cells and reduces the risk of losing rare populations.
2) Target Antigen
Low-expressing or sensitive antigens: The "Stain-Lyse-Wash" protocol is recommended to protect antigen epitopes from potential damage by the lysis buffer.
Common cell populations or large-scale multi-indicator panel: The "Lyse-Stain-Wash" approach is ideal for high-efficiency antibody binding and maximising throughput.
3) Experiment Scale and Throughput
Small-scale experiments (less than 10 tubes): The "Stain-Lyse-Wash" protocol is quicker and more straightforward.
Large-scale experiments (more than 10 tubes): The "Lyse-Stain-Wash" protocol allows for more streamlined processing and higher throughput.
4) Data Quality and Reproducibility
Small-scale experiments: The "Stain-Lyse-Wash" protocol is easier to standardise, reducing operational variability.
Large-scale experiments: The "Lyse-Stain-Wash" protocol ensures better consistency across tubes, making it easier to compare results and improve reproducibility.
4. Conclusion
The decision of whether to stain or lyse first is not about choosing right or wrong, but about making the right trade-off based on experimental needs. Every step in flow cytometry can influence the results, and understanding the rationale behind each approach helps in choosing the one that best aligns with your research goals. Ultimately, attention to detail and careful choice of protocol will ensure that your flow cytometry experiments deliver reliable and meaningful data.
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