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Looking for Ways to Streamline Your Cell-Based Assays Using Fresh or Frozen Leukopaks?

Small Scientist on July 27, 2021

The use of human primary cells has led to major breakthroughs in drug discovery research, cancer research and cell therapy research. As an essential source of primary immune cells, leukopaks are an ideal starting material for downstream cell isolation when large numbers of cells are required. Watch the on-demand webinar Leukopak Processing: Tips & Tricks for Streamlined Cell Isolation presented by STEMCELL Technologies and gain insights and techniques for efficient leukopak sample processing to reduce the time and reagents required to process cells of interest. If you’re short on time, we asked STEMCELL to share the top-ten most frequently asked questions about leukopak processing.





1. Why are leukopaks red, and are non-red leukopaks available?
Leukopaks are red due to residual red blood cells (RBCs) in the sample; however, they do not affect the cell quality. Non-red leukopaks are not available as the degree of redness is often impacted by donor-to-donor variability. For a usual leukopak, we see a 3:1 ratio of RBCs to WBCs. Compared to that of whole blood (700:1), we can see that leukopaks are a highly enriched source of WBCs and that there are minimal RBCs in the bag.

2. How many cells are available in a fresh leukopak?
The cell count varies depending upon the donor. As a result, the number of cells that can be isolated after processing the sample cannot be guaranteed. However, here are STEMCELL Technologies’ minimum cell counts* for product release at the time of shipping:

Full size leukopak: 9 x 10e9
Half-size leukopak: 5 x 10e9
Quarter-size leukopak: 2.5 x 10e9
Tenth-size leukopak: 0.9 x 10e9

*These minimum cell counts are the acceptance criteria for product release at the time of shipping. Minimum cell counts are the same for frozen leukopaks; however, for frozen leukopaks, cells are counted at the time of freezing. Viability of these cells as well as the fresh cells after washing is typically above 90%.

3. How long can a fresh leukopak be stored at 4°C or room temperature (RT)?
We do not recommend storing leukopaks at room temperature (RT). If storage upon arrival is needed for longer periods of time, storing the leukopak at 4°C has been shown to be acceptable depending on your cell type of interest.

4. Is there any option of automated cell isolation for the full fresh or frozen leukopak?
Yes, there are multiple automation options offered by STEMCELL, such as the RoboSep™-16 and the RoboSep™-C for fully automated cell isolation from both fresh and frozen leukopaks.

5. What is the percentage of viable cells in a frozen leukopak?
Upon cryopreservation, cell viability in the frozen leukopak is greater than 90%. After thawing and the cleanup process, the viability is usually greater than 90%.

6. What dilution do you recommend to process a leukopak by density centrifugation?
Diluting the leukopak to a 1:4 ratio with PBS + 2% FBS works well for Lymphoprep™ or Ficoll™ processing. Due to the concentrated nature of leukopaks, the cell layer at the interface can be quite thick. Lower dilution will result in a thicker cell layer at the interface and may prohibit efficient collection of cells.

7. Is there a risk of clotting after washing off the cryopreservation reagents and anticoagulant?
If washed with DNAse and FBS-containing media, there is minimal risk of clumping after the anticoagulant and cryopreservation media are washed off.

8. After isolating cells from the leukopak, what freezing medium would you recommend to ensure optimal quality of my cells upon thaw?
We recommend using CryoStor® CS10 to freeze samples as this is a serum- and animal component- free product that will help your cells handle the freeze-thaw process. While other freezing media may be used, there may be varying degrees of success based on the formulation.

9. Are there any advantages of working with frozen leukopaks compared to fresh leukopaks?
Both fresh and frozen leukopaks are ideal starting materials for downstream cell isolation when large numbers of cells are required, reducing the time and reagents needed to process cells of interest. However, frozen leukopaks provide additional convenience by allowing you to start experiments as per your schedule. Additionally, cell viability may be better preserved in frozen leukopaks during global shipping. Frozen leukopaks can also provide you with access to samples from the same lot at a future date.

10. Can I request which medications donors are or are not using? Can donors be recalled for my experiments?
You may request donors with specific medication and other requirements. However, this may affect lead time and could also require a custom quote in certain situations. Donors can be recalled for your experiments; however, future donations are at the discretion of the donor and are not guaranteed. Generally, leukopak donors are recallable 56 days after their last donation. Please contact us at techsupport@stemcell.com for more information.

For more frequently asked questions and answers on leukopaks or to learn more about our primary cell offering, read primary cells FAQs.

About STEMCELL Technologies
At STEMCELL Technologies, our mission is to advance scientific knowledge globally by providing high-quality, innovative reagents, tools, and services that enable scientific research. As Scientists Helping Scientists, we’re here to support your research endeavors with our ethically sourced, high-quality fresh and frozen human primary cells, as well as cell isolation and cell culture products.