Membrane proteins are major sensors of extracellular stimuli and initiators of intracellular signal transduction, and their abundance on the cell surface in particular is often dynamically regulated even when there are no significant changes of their total abundance in a cell. This protocol is designed to biochemically label and separate membrane proteins on the plasma membrane from those in the intracellular compartments. In conjunction with co-immunoprecipitation and western blot analysis, functional analysis of dynamic interaction of membrane proteins with other membrane proteins or intracellular adaptor and effector proteins can be achieved.

Antigen recognition and activation of T cell receptor (TCR) triggers transient calcium release from intracellular compartments and subsequent sustained calcium influx through cell surface Icrac channels. Sustained elevation of the cytoplasmic calcium level activates many calcium-dependent enzymes and transcription factors, which are essential for T cell activation and function. This protocol uses non-ratiometric dyes, in combination with flow cytometry, to monitor TCR-triggered calcium changes over time, and is a simple assay to examine the existence of T cell calcium mobilization defects in transgenic mice.

Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans.An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that lead to renal impairment in SLE and other renal inflammatory diseases.

This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.