Bio Spec Overview
Ignite Creation Date:
2025-12-24 @ 2:24 PM
Last Modification Date:
2025-12-24 @ 2:24 PM
Retention:
SAMPLES_WITHOUT_DNA
Description:
For the ERK activation assay, about 5 mL of whole blood is layered onto 3 mL Histopaque in a 15 mL centrifuge tube, and centrifuged. The lymphocyte-containing cell layer is removed and transferred for washing. After a second wash, cells are resuspended and rested. They are then stimulated by addition of phorbol myristate acetate and sample aliquots are removed at short intervals, fixed and permeabilized. Fixed, permeabilized cells are stained by addition of Alexafluor488-labeled monoclonal antibody to phospho-ERK in the dark for 30 min. Washed, resuspended cells are analyzed in a Coulter flow cytometer.
Section:
For the ERK activation assay, about 5 mL of whole blood is layered onto 3 mL Histopaque in a 15 mL centrifuge tube, and centrifuged. The lymphocyte-containing cell layer is removed and transferred for washing. After a second wash, cells are resuspended and rested. They are then stimulated by addition of phorbol myristate acetate and sample aliquots are removed at short intervals, fixed and permeabilized. Fixed, permeabilized cells are stained by addition of Alexafluor488-labeled monoclonal antibody to phospho-ERK in the dark for 30 min. Washed, resuspended cells are analyzed in a Coulter flow cytometer.