High-Throughput Immunodensity-Based Cell Subset Isolation Directly from Whole Blood
- Document # 28726
- Version 2.1.0
- Dec 2024
Case Study: High-Throughput Isolation of Natural Killer Cells for the Assessment of In Vitro Cytotoxicity Using the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 System
In order to streamline the workflow of immune cell separation directly from whole blood samples and achieve high-throughput sample processing, Dr. Ajay Jain鈥檚 lab at the University of Maryland School of Medicine adopted a fast and efficient natural killer cell isolation method鈥攖he 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system. This technical bulletin discusses how this rapid cell isolation system, which relies on immunodensity, reduced the time to obtain cells from 450 mL of blood from four hours to a single hour without compromising cell function or performance in downstream assays.
Background
The Need For High-Throughput Cell Isolation
Immune cell isolation plays an important role in areas such as drug discovery and development, vaccine research, and translational immunology. For example, isolating immune cells is routinely necessary to evaluate the efficacy of immunostimulatory compounds, assess the immunogenicity of vaccine candidates, or investigate interactions between the immune system and infectious agents.1-4
The movement towards more physiologically relevant assays based on primary human cells has created the need for a fast and efficient method of isolating immune cells from large numbers of whole blood samples.2,4 To facilitate this type of high-throughput cell processing, 海角破解版 Technologies developed the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system that rapidly isolates cells directly from whole blood in as little as 25 minutes.
The 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system combines a unique immunodensity cell isolation reagent (搁辞蝉别迟迟别厂别辫鈩) with a specialized cell processing tube (SepMate鈩) to reduce the number of steps needed for cell isolation. This saves time, minimizes variability between users, and allows for efficient, high-throughput sample processing. Using this cell isolation system results in untouched and highly purified cells without the need for columns or immunomagnetic beads. Cells are immediately available for use in a variety of downstream assays, such as cytotoxicity testing, compound screening, and other applications where it is important to obtain viable, functional cells with minimal manipulation.
Evaluation of the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 System
Traditional methods for isolating lymphocytes from blood are laborious and time-consuming, and require technical expertise and precision. These methods typically involve isolating peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation (DGC) before enriching specific cell subpopulations using immunomagnetic column-based systems. At the University of Maryland School of Medicine, Dr. Ajay Jain and colleagues routinely isolate natural killer (NK) cells from large numbers of human samples. Isolating NK cells from a 450 mL unit of blood by DGC followed by immunomagnetic column-based selection can take up to 4 hours. In addition, following this method makes it difficult to process multiple samples quickly and efficiently.5 In order to find solutions to streamline their workflow and achieve higher- throughput sample processing, Jain鈥檚 lab evaluated the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system and assessed its impact on NK cell marker expression and cell cytotoxicity.
Methods
Dr. Jain鈥檚 group assessed and compared the in vitro cytotoxicity of NK cells that were isolated from 450mL of whole blood using two different methods: DGC followed by column-based immunomagnetic cell isolation or the 搁辞蝉别迟迟别厂别辫鈩 Human NK Cell Enrichment Cocktail and SepMate鈩 cell isolation system (Catalog #15065 and Catalog #85450), as previously described.5 NK cellular cytotoxicity was then assessed against HT29 colon cancer and K562 leukemia target cells using a chromium release assay. Expression of NK cell activation markers was assessed using flow cytometry.
Results
Dr. Jain鈥檚 group found that isolation of NK cells was significantly faster with the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system: a 450 mL unit of blood could be split into multiple samples and processed in a single hour. Comparitavely, NK cell isolation by DGC followed by column-based immunomagnetic selection (DGC/immunomagnetic selection) took up to four hours.5
NK cells isolated by 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 were highly purified (86.7% as assessed by flow cytometry) with intact cytolytic function. To quantify the cytolytic activity of effector cells, Dr. Jain鈥檚 group used an in vitro assay to assess the capacity of NK cells to kill target HT29 colon cancer cells (Figure 1) and K562 leukemia cells (not shown here). HT29 cells express high levels of the epidermal growth factor receptor (EGFR) and are susceptible to NK-mediated antibody-dependent cellular cytotoxicity in the presence of anti-EGFR antibody cetuximab.6 The results showed that NK cells isolated with the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system retain the capacity to kill HT29 colon cancer cells by antibody-dependent cellular cytotoxicity (Figure 1) and K562 leukemia cells by direct cell-mediated killing (not shown here).5 Flow cytometric evaluation of NK cell activation markers demonstrated that NK cells isolated using 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 have similar expression profiles to cells isolated using DGC/immunomagnetic selection (Figure 2).5
Figure 1. NK Cells Isolated with 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 Exhibit In Vitro Cytotoxicity.
NK cell in vitro cytotoxicity against HT29 colon cancer cells was assessed with cells isolated by DGC followed by immunomagnetic selection (left panel) or 搁辞蝉别迟迟别厂别辫鈩 Human NK Cell Enrichment Cocktail (Catalog #15065) and SepMate鈩 (Catalog #85450) (right panel). In vitro conditions included culture with media alone (circles), 10 渭g/mL human IgG1 isotype control (diamonds), or 10 渭g/mL cetuximab (triangles). The figure shows the results of one representative experiment. Figure courtesy of Dr. Ajay Jain.
Figure 2. Surface expression of NK cell activation markers as evaluated by flow cytometry.
NK cells were isolated by DGC followed by immunomagnetic selection (DGC/Immunomagnetic selection), 搁辞蝉别迟迟别厂别辫鈩 Human NK Cell Enrichment Cocktail (Catalog #15065) and SepMate鈩 (Catalog #85450), or 搁辞蝉别迟迟别厂别辫鈩 Human NK Cell Enrichment Cocktail (Catalog #15065) and SepMate鈩 (Catalog #85450) followed by red blood cell (RBC) lysis. The surface and intracellular expression of NK cell activation markers was evaluated by flow cytometry. Expression is shown as mean fluorescent intensity (MFI). The expression of surface and intracellular activation markers, aside from CD16, is comparable between NK cells isolated by DGC/immunomagnetic selection and NK cells isolated with 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 in the absence of RBC lysis. Figure courtesy of Dr. Ajay Jain.
"The SepMate鈩 tubes鈥 provide a mechanical barrier between the purified effector cells and the density gradient medium. The desired cell populations remain above the barrier and can be poured out of the tube with no technical expertise. This method allows for the concurrent rapid purification of NK cells and CD8+ and CD4+ T cells from multiple human donors, which makes downstream applications (i.e. flow cytometry, ELISA, assessment of in vitro cytotoxicity) more practical to perform in a high-throughput manner."
So EC et al. (2013)
Conclusions
The 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system allows efficient negative isolation of cell subsets directly from whole blood in a quarter of the time compared to density gradient centrifugation followed by column-based immunomagnetic selection. The 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system requires no technical expertise, facilitates rapid cell isolation from many samples at once, and yields fully functional cells for highthroughput downstream assays. For more information on the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 system and how it works, see page 3.
How the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 System Works
搁辞蝉别迟迟别厂别辫鈩
搁辞蝉别迟迟别厂别辫鈩 isolates highly purified cells directly from blood by crosslinking unwanted cells to red blood cells, forming immunorosettes. These immunorosettes pellet during density gradient centrifugation, leaving untouched and highly purified target cells at the interface between the plasma and the density gradient medium.
The SepMate鈩 Advantage
Using the SepMate鈩 tube makes the 搁辞蝉别迟迟别厂别辫鈩 procedure even faster and easier. The SepMate鈩 insert creates a physical barrier between the sample and density gradient medium, allowing the sample to be rapidly pipetted or poured into the tube. Centrifugation time is reduced to just 10 minutes, and purified target cells can simply be poured into a new tube. SepMate鈩 can also be used on its own for hassle-free PBMC isolation from whole blood in just 15 minutes.
Why Use the 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 System?
- Isolate untouched cell subsets from whole blood during your standard density gradient centrifugation step in as little as 25 minutes.
- Choose from a wide range of 搁辞蝉别迟迟别厂别辫鈩 reagents to isolate the cell subset of your interest.
- Obtain highly purified, unlabeled, viable, and functional cells.
- Eliminate errors with an easy-to-use system that does not require columns, magnets, or other special equipment.
- Combine 搁辞蝉别迟迟别厂别辫鈩 with SepMate鈩 to minimize variability between separation, or use 搁辞蝉别迟迟别厂别辫鈩 on its own.
The 搁辞蝉别迟迟别厂别辫鈩 and SepMate鈩 Procedure
Watch a video showing how to combine SepMate鈩 with 搁辞蝉别迟迟别厂别辫鈩 for fast cell subset isolation directly from whole blood.
Product Listing
Select 搁辞蝉别迟迟别厂别辫鈩 Products for Cell Isolation by Negative Selection
Select 搁辞蝉别迟迟别厂别辫鈩 Products Cell Depletion Cocktails
SepMate鈩 Products
1. SepMate鈩 (IVD) is available in Australia, Canada, Europe, the United Kingdom, and the United States of America, as an In Vitro Diagnostic (IVD) device for the isolation of mononuclear cells from human whole blood and bone marrow by density gradient centrifugation. This product is also available in China where it is considered a non-medical device by the China Food and Drug Administration (CFDA), and should therefore be used as general laboratory equipment.
2. SepMate鈩 for Research Use Only (RUO) is available in regions where SepMate鈩 is not
registered as an IVD device.
Fresh Human Peripheral Blood Products
CP2D2
EDTA2
Na Heparin2
1. Fresh products (excluding bone marrow), currently available in the United States and Canada
(excluding Quebec). Fresh bone marrow currently available in the United States only. Please
contact Product and Scientific Support (techsupport@stemcell.com) for further information.
2. ACDA - Acid Citrate Dextrose Solution A; CP2D - Citrate-Phosphate-Double Dextrose; EDTA -
Ethylenediaminetetraacetic Acid; Na Heparin - Sodium Heparin
Density Media
1. 尝测尘辫丑辞辫谤别辫鈩 has the same density as Ficoll-Paque庐 and can be substituted for Ficoll-Paque庐 without any need to change your existing protocols.
References
- World Health Organization. (2005) Technical Report Series, No. 927. Clin Transplant 18(5): 558鈥563.
- Slater K. (2001) Curr Opin Biotechnol 12(1): 70鈥4.
- McMillin DW et al. (2012) Blood 119(15): e131鈥8.
- S谩ez-Ciri贸n A et al. (2010) Nat Protoc 5(6):1033鈥41.
- So EC et al. (2013) J Immunol Methods 394(1鈥2): 40鈥8.
- Schlaeth M et al. (2010) Cancer Sci 101(5): 1080鈥8.
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