Leukopak PBMC Isolation With Akadeum’s Microbubbles

Importance of PBMC Isolation From Leukopaks

Human peripheral blood mononuclear cells (PBMCs) include lymphocytes, monocytes, and dendritic cells. They t develop from stem cells within the bone marrow. PBMCs play a vital role in the innate and adaptive immune system. Due to this, PBMCs are at the center of many scientists’ research studies. To obtain a large quantity of PBMCs,  scientists often order leukopaks which are collected via leukapheresis and can supply scientists with up to 20 billion PBMCs per leukopak. 

What Is a Leukapheresis Machine?

A leukapheresis machine is a specialized apheresis device that extracts leukocytes from the bloodstream and returns most other blood components—plasma and red blood cells (RBCs)—to the donor. The resulting blood-derived product is highly concentrated with PBMCs from a single donor and is distributed in the form of leukopak. 

Using centrifugation, the leukapheresis procedure filters through the cells to obtain the highest concentration of white blood cells with the smallest concentration of RBC and platelet contamination. This is due to the specific gravity of the individual blood cells, with red blood cells having the highest specific density, allowing the cells to be separated over numerous centrifugation steps. 

This process seems simple enough, but the technology necessary to spin and separate the blood components while maintaining the flow pressure, volume, and speed of the donor’s bloodstream are difficult to calibrate. Although the protocol is highly reliable and regimented, there is often great variability in the amount of RBC and platelet contamination in the final leukopak product. 

Leukopaks are commonly processed further to isolate the individual cell subsets of interest suspended within the leukopak material. The remaining RBCs and platelets are removed from the cells of interest using a cell separation method, commonly a density gradient. Proper leukopak processing is imperative to immunology studies. Using Akadeum innovations, research moves swiftly between protocol steps, including otherwise complex undertakings like T-cell isolation. Using microbubbles, T cells can be isolated with ease for high-quality downstream analysis. 

Microbubble Leukopak PBMC Isolation Protocol

Bypass a cumbersome RBC depletion step and avoid possible damage inflicted upon cells of interest by using Akadeum’s BACS™ protocol. The Akadeum BACS™ protocol is robust and can withstand some RBC contamination, all while utilizing negative selection to keep the cells of interest untouched while removing unwanted cell types. 

With microbubble technology, there is no need to isolate PBMCs from the leukopak before processing, allowing whole leukopaks to be processed simultaneously. 

Materials needed:

• 50 mL conical centrifuge tubes
• Separation centrifuge tubes
• Leukopak (thawed from cryopreserved or fresh)
• Antibody cocktail
• Separation buffer
• BACS™ microbubbles

Aliquot Leukopak

 The leukopak contents are first transferred evenly into as many 50 mL conical tubes as necessary. Each conical tube is then adjusted to 45 mL using the provided Separation Buffer and centrifuged to wash the contents.   The sample is then washed a second time using a Separation Buffer.

Transfer to Separation Tubes

Transfer the washed contents into Akadeum’s novel microbubble separation tube. Label unwanted cells, such as RBCs, with our specially-tailored antibody cocktail. Allow the antibodies to bind to unwanted cell populations. This will prep the cells for microbubble attachment in the following steps.

Add BACS™ Microbubbles

After incubation, add BACS™ microbubbles to the separation tube, mix thoroughly, and incubate to allow the microbubbles to bind to the unwanted cells. 

Centrifuge Samples

Centrifuge the separation tube to allow unbound cells of interest to fall to the bottom of the sample. The buoyancy of the microbubbles fights against the downward force, keeping unwanted cells on the sample’s surface. 

Drain Cells of Interest Into a Fresh Tube

Utilizing the separation tube, easily drain the cells of interest into a fresh tube away from the microbubbles and unwanted cells. By separating using gravitational pull and a separation tube, the cells are in their most untouched state for further downstream applications. 

How Akadeum’s BACS™ Microbubble Leukopak Cell Isolation Process Is Different

Very few blood separation protocols allow the complete processing of a leukopak using the unprocessed leukopak material. This versatility enables ease of protocol, minimizing time spent and resources used. The main benefit of the BACS™ system is that T cells can be isolated straight from the leukopak, eliminating the need for time-consuming PBMC isolation or density gradient centrifugation steps. 

Our gentle and efficient system addresses typical cell separation issues and allows researchers to focus on the experimental applications of the cells of interest. Without magnetics, countless washes, and tube transfers, it’s easy to see why the BACS™ protocol is convenient and reliable. 

Obtain a higher yield of cells in under an hour and experience for the first time how streamlined your lab workflow can be. Built to aid in scalability, scientists can depend on Akadeum’s process.

BACS™ Is Gentle On Fragile Cell Populations

Cell separation protocols often include multiple magnetic incubation, washing, centrifuging, and volume-transferring steps. In addition to being laborious, these processes are sometimes damaging to desired cell populations and can endanger the success of the cells’ downstream applications. 

Traditionally performed using a Ficoll density gradient, the blood-derived material would undergo several mixing, layering, and centrifugation steps. This unforgiving manual separation does not guarantee a pure quality sample and is generally too harsh and time-consuming to meet modern research needs.

Contact Akadeum to Learn More About Our Technology

Explore how Akadeum can maximize your workflow and increase the quality of your cell isolations. Access high concentrations of T cells in just minutes using our Microbubble Leukopak Human T Cell Isolation KitHuman T Cell Isolation Kit.