Example Studies of T Cell Characterization Available at Reaction Biology
Tracking of T cell division via CFSE after activation via plate-bound anti-CD3 antibody
T cell proliferation is one of the hallmarks of successfully activated T cells. Carboxyfluorescein succinimidyl ester (CFSE) is a cell-permeable dye that binds covalently to intracellular molecules and is retained within cells for very long periods. The intensity of CFSE in cells can be measured by flow cytometry. With each cell division, the intensity halves, enabling the identification of proliferating cells.
PBMCs are plated in multi-well plates coved with anti-CD3 antibodies, which engage the T cell receptor, triggering T cell activation and expansion. CFSE dye is used to track cell division by measuring CFSE intensity in the cells.
Healthy donor PMBCs were labeled with CFSE and either cultured in the presence of two different concentrations of plate-bound anti CD3 (OKT3) or left untreated. After 5 days, cells were harvested, stained with a fixable viability dye and surface markers (CD3, CD4, and CD8), and fixed with 4% PFA. Cells were acquired using a Cytoflex S (Beckman Coulter).
Peripheral blood mononuclear cells (PBMCs) are a mixture of immune cell populations developed in the bone marrow from hematopoietic stem cells. The PBMCs include particularly those with a single round nucleus, composed mainly of lymphocytes, dendritic cells, and monocytes. We isolate PBMCs out of buffy coats, which entail the entire cellular fraction of one blood donation unit.
The material is ideal for the isolation of a large number of immune cells from one healthy donor and allows us to have enough material to isolate rare cell types (like NK cells or monocytes) and to have several frozen vials from the same donor in order to perform different tests with the same cell batch if needed.
Since the frequency of different cell types within PBMCs varies from donor to donor, we performed a broad characterization of the main immune cell populations. With this information, we can choose the best donor for a project depending on the particular need for a certain immune cell population.
Healthy donor PBMC were isolated from buffy coats after density gradient centrifugation and stored in liquid nitrogen until further use. PBMC were thawed, labeled with a fixable viability dye, and stained for surface markers for T cells (CD3, CD4, CD8), monocytes (CD14), B cells (CD19), and NK cells (CD56).
We also included a Human Leucocyte Antigen Serotype (HLA-A2) marker and stained for HLA-DR, which is expressed on the surface of antigen-presenting cells (macrophages/monocytes, B cells, and dendritic cells) and is upregulated in response to stimuli.
|Immune cell population||Frequency of viable cells (% of viable single cells)|
|CD3+ CD4+||16 %|
|CD3+ CD8+||10 %|
|CD3- CD19+||6 %|
|CD3- CD56+||11 %|
|CD3- CD14+||39 %|
The frequencies of several immune cell populations in the PBMCs of one donor are shown. Please reach out to inquire about the profiles of other PMBC donors or sources to ensure we align the experiment setup with your research questions.