What kinds of stimulants are used for flow calcium assays?

There are various types of stimulants used for flow calcium assays. Some common examples include ionomycin, phorbol myristate acetate (PMA) and thapsigargin. Ionomycin serves as a positive control in experiments to generate the maximum calcium flux ratio. This ionophore has been utilized at concentrations between 10^(-7) to 10^(-5) M and also facilitates the movement of calcium from intracellular stores to the cytosol. PMA works by activating protein kinase C (PKC), which in turn activates Ras, a small signaling protein. When PMA and ionomycin are used together, they can activate signaling pathways downstream of PKC and Ras without triggering specific events initiated by T cell receptors (TCR). This allows researchers to selectively stimulate certain cellular responses for study without involving the entire TCR signaling process. Calcium influx is one of the crucial signaling processes upon stimulation of TCRs. Thapsigargin is a compound that inhibits the SERCA pump, a protein responsible for transporting calcium ions into the endoplasmic reticulum (ER). By blocking this pump, thapsigargin induces the depletion of calcium stores within the ER. As a result, there is a moderate increase in cytosolic calcium levels.