Understanding B cell adaptive immunity with real-time live-cell analysis

B cells are at the center of the adaptive immune system and are responsible for the production of antigen-specific immunoglobulin directed against invasive pathogens. B cell activation and proliferation are crucial for the secretion of antibodies, but continuous stimulation can also lead to the survival and growth of B cell leukemia’s and lymphomas, such as Hodgkin’s and non-Hodgkin’s lymphomas.

The IncuCyte® live-cell analysis system has been used to evaluate key functions of B cells, including activation & clustering and antibody internalization assays, as well as evaluating cell death of B cell cancers via apoptosis and immune cell killing assays.


Activation & Clustering

Proliferation of a non-adherent B-cell line. The proliferation of WIL-2NS cells, a B-cell line, quantified from phase contrast images.  As expected the starting confluence is seeding density dependent.  Validation of the confluence measurement was achieved by seeding known amounts of cells per well, allowing settling and then quantifying using the phase contrast algorithm.  In addition, cells were subsequently permeablized and the ATP content determined to provide a secondary readout.  A very strong correlation between % confluence and cell number or ATP determination was observed.

Apoptosis (Annexin V Green)

Measure apoptosis of B cells following cytotoxic challenge. Representative images of WIL2-NS B lymphoma cells taken 24h after exposure to either vehicle or camptothecin (1 µM) in the presence of IncuCyte® Annexin V Green reagent.  The time-course depicts the increase in green fluorescence emanating from the IncuCyte® Annexin V Green reagent, indicating a time- and concentration-dependent increase in externalized phosphatidylserine induced by camptothecin.

Immune cell killing

Measure ADCC of leukemic cells. Blended phase and fluorescent images of Ramos B lymphocytes expressing NucLight Red® nuclear marker in the presence immune cells, either untreated or in the presence of  IL-2 (10 ng/ml) & IL-12 (10 ng/mL) or Rituxan (15 ng/mL – 100 ug/mL). Cytotoxicity was quantified based on the loss of red fluorescent intensity.

Quantifying antibody internalization

Internalisation of the clinically used antibody Rituxan. Raji (B cell like) cells (30K/well) were treated with IncuCyte® FabFluor labeled Rituxan (10 ng/mL to 10 µg/mL). HD phase and red fluorescence images were captured every 30 minutes using a 20x objective over 12 hours. All data shown as a mean of at least 4 wells ± SEM, time course data shown as normalized red area (red area/phase area).