Analysis of Natural Killer (NK) Cells via Flow Cytometry: Function, Isolation, and Clinical Relevance

Natural killer (NK) cells represent one of the three groups of lymphocytes, alongside T and B cells. Unlike the latter, they belong to the innate immune system and constitute the first line of defense against a wide range of pathogens. Simply put, their function is to destroy cells—whether they are infected by a pathogen or potentially harmful cancerous cells.

The Dual Function of NK Cells

NK cells are naturally programmed to detect and eliminate infected or cancerous cells that express specific markers of malignancy, while “sparing” healthy cells that do not express such markers.

However, certain alterations, such as mutations, can impair these cells’ ability to distinguish between healthy and malignant cells. Instead, they may indiscriminately “kill” all cells, leading to severe autoimmune diseases where the body attacks itself.

Analysis Protocol

To analyze the profile of NK cells, the following steps must be followed:

1. PBMC Isolation

• Approximately 4 mL of human blood must be collected  and diluted 1:1 with serum.

• 4 mL of separation medium is then added to the sample.
• The mixture is to be centrifuged at 650xG for 24 minutes, and the formation of layers is observed. The thin middle layer, corresponding to PBMCs (including NK cells), needs to be collected and placed into a new tube containing serum.
• The previous step must be repeated. 
• To isolate NK cells, FBS and separation medium are added to the mixture. Following this, the sample is centrifuged at 1200xG for 10 minutes. The supernatant must then be removed, and the sediment is resuspended in serum.. 

2. Employ specific markers for NK cells and analyze them using flow cytometry.. 

For the centrifugation process, the Digicen 22 and Unicen 21 centrifuges are ideal due to their advanced features and versatility. The Digicen 22 provides digital control of speed and time, enabling precise adjustments of centrifugal forces with high reproducibility. Additionally, it features the REI System (Rotor Easy to Install), facilitating quick and easy rotor exchange. On the other hand, the compact and robust design of the Unicen 21 makes it an excellent choice for laboratories prioritizing efficiency and ease of use.

Determining the toxicity profile of NK cells in the context of autoimmune diseases is of great importance for establishing treatment strategies. This can be achieved using the above-described protocol, where centrifugation plays a critical role.