Recursos Técnicos - Posters Científicos
Stimulation of Human Peripheral Blood Mononuclear CellsDescargar
January 24, 2020
Author: Paul Held, BioTek Instruments, Inc., Winooski, VT
Human Peripheral Blood Mononuclear Cells (PBMCs) are routinely isolated from blood samples collected from individuals and then used in several fields of research including autoimmune disorders, infectious diseases, vaccine development and cancers. The response of this diverse group of cells to different stimuli offers insights into their role in disease and the development of treatment modalities.
The ELISPOT assay procedure is very similar to that of a conventional ELISA, with the exception that ELISPOT assays use specialized plates that have a porous PVDF membrane rather than conventional plastics. This membrane captures specific secreted molecules. The analyte remains in close proximity to the location where the secreting cell was situated. The resulting spots are used as means to count the number of activated cells in a population. Traditionally ELISPOT assays have been quantitated either manually by physically counting the spots under a stereoscope or automated using a dedicated ELISPOT counter.
Here we describe the use the Cytation™ 7 Cell Imaging Multi-Mode Reader in conjunction with Gen5™ software to quantitate changes in cytokine secretion in PBMCs using the colorimetric ELISPOT assay format. By automatically counting the developed spots from an ELISPOT assay, phorbol 12-myristate 13-acetate (PMA) was demonstrated to stimulate (EC50=0.28 ng/mL) the secretion of IL-2 and IFN-γ in PBMCs in a dose dependent fashion. Additionally, the compound triptolide was shown to inhibit (EC50=30 nM) the stimulatory effects of PMA. Assay optimization data including cell number, stimulant pharmacology, and inhibitor results will be presented. Data includes the use of simultaneous two-color spot counting for multi-analyte determination. In addition to PDVF membranes, ELISPOT assays can be performed using silver deposition on clear polystyrene plates. A comparison of bright field imaging with the inverted camera and transmitted light imaging with the upright camera demonstrates the equivalence of the two methods.