Detecting Cytokines in Growth Media
We study cytokine production of immune cells isolated from peripheral blood given by healthy donors and patients. After collecting cell supernatants from Peripheral Blood Mononuclear Cells (PBMCs), the media is frozen at -80°C until protein analysis. Each healthy donor and patient is tested for cytokine production in response to Candida albicans, Glucan (fungal cell wall) and Pam3CSK4 (a synthetic lipopeptide). We purchased the MESO QuickPlex SQ 120 in November 2020 with a grant from NIH to better study the production of cytokines from PBMCs.
"PBMCs are a variety of specialized immune cells that work together to protect our bodies from harmful pathogens. Their location in peripheral blood is significant because they act as a line of defense from infection and disease." - What Is PBMC?
The MESO QuickPlex SQ 120 is a compact system that offers high-performance ECL immunoassays to detect cytokines down to less than a picogram. We are interested in the TH17 pathway which detects pathogens and sends chemical signals to neutrophils and macrophages to attack. The kit that we use detects all ten of the following cytokines: IFN-γ, IL-1β, IL-6, IL-10, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, TNF-α.
Top and bottom view of 96-well ECL plate
"Electrochemiluminescent labels generate light when stimulated by electricity in the appropriate chemical environment. This reaction is incorporated into our immunoassays to provide the light signal used to measure important proteins and other biomedical molecules." - Why Electrochemiluminescence?
MESO QuickPlex SQ 120 at ETSU
(my cell phone camera isn't great lol)
Detection of IL-1ß by ELISA
We also use Enzyme-Linked Immunosorbent Assays (ELISAs) for IL-1ß using a Human IL-1beta ELISA kit with pre-coated plates. I used to coat my own plates at UNC Charlotte! Not only is the pre-coated plate more reliable in regards to reproducibility, they are a huge time saver. This assay is super easy to do as well. This isn't rocket science. lol
"IL-1β is a potent immuno-modulator which mediates a wide range of immune and inflammatory responses including the activation of B and T cells. IL-1β is expressed in several cell types, including monocytes, tissue macrophages, Langerhan cells, dendritic cells, T lymphocytes, B lymphocytes, and natural killer cells. IL-1β plays a role in many physiological and pathological conditions including rheumatoid arthritis and cancer." - LEGEND MAX™ Human IL-1β ELISA Kit
Why Do We Study Cytokines?
By measuring cytokines or chemical messages that cells use to talk to each other, we can better understand the immune response by healthy individuals and sick patients. By comparing these responses among the healthy aging population and with sepsis and infectious disease patients, we will better understand aging of immune cells and exacerbated immune reactions such as those seen with sepsis. In sepsis, the immune response is a severe and persistent inflammatory reaction.
"Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action). There are both pro-inflammatory cytokines and anti-inflammatory cytokines. There is significant evidence showing that certain cytokines/chemokines are involved in not only the initiation but also the persistence of pathologic pain by directly activating nociceptive sensory neurons." - Cytokines, Inflammation and Pain
Have a great weekend!!! Peace 😇