Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Summary

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Abstract

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Introduction

Calcium is an important second messenger responsible for the transmission of cellular physiological and pathophysiological signals¹. Cytosolic calcium concentrations are kept low through the activity of calcium pumps and calcium exchangers. The sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) refills the endoplasmic reticulum (ER) calcium store as part of a “pump leak” system whereas the plasma membrane calcium ATPase (PMCA) extrudes calcium to the extracellular compartment, both in ATP-dependent manners². Physiological messengers, such as hormones or neurotransmitters, transmit their signals through G-protein coupled recepto....

Protocol

1. Preparation of Reagents and Solutions

1. Prepare a modified Hank’s balanced salt solution (HBSS) (in mM: 136.9 NaCl, 5.37 KCl, 0.34 Na₂HPO₄, 0.44 KH₂PO₄, 4.17 NaHCO₃, pH 7.2, no phenol red). Store at 4 °C.
2. Make 1.5 M CaCl₂ and 2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) stock solutions using distilled water.
3. Prepare a 250 mM stock solution of probenecid. For the free acid form (often referred to as water insoluble), dissolve probenecid powder in 1 N NaOH, fill to three quarters of the final volume with distilled water and slowly titr....

Results

To increase cytosolic calcium, two pharmacological compounds were applied to renal proximal tubule cells (WKPT-0293 Cl.2) loaded with cell permeable calcium binding dye, Fluo-3-AM. Nontreated control samples were loaded with calcium binding dye in the presence of probenecid and were mixed but without the addition of any compounds. Thapsigargin (TG) is a classical inhibitor of the SERCA pump leading to leakage out of the ER and resulting in increased cytosolic calcium. Tunicamycin (TUN) blocks N-glycosylation of proteins .......

Discussion

Calcium is a key event in multiple cellular processes acting as a second messenger signaling molecule and also as a means to communicate between the ER and mitochondria. The ability to measure changes in free cytosolic calcium concentrations is a useful technique that can apply to all areas of cell biology. There are various methods to detect cytosolic calcium signals. Classically, signals from a ratiometric calcium indicator, such as Fura-2, are monitored in live cells using a fluorescence imaging setup and absolute cal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluo-3, AM	Invitrogen	F-1241	Cell permeable
Probenecid	Sigma-Aldrich	P-8761	Water insoluble, dissolve in 1 N NaOH
0.05% Trypsin-EDTA (1X)	Invitrogen	25300-062
Ionomycin	Sigma-Aldrich	I9657
Thapsigargin	Tocris Bioscience	1138
Tunicamycin	Sigma-Aldrich	T7765
FACSCalibur Flow Cytometer + CELLQuest software	Becton Dickinson
Windows Multiple Document Interface for Flow Cytometry (WinMDI)	Joe Trotter, The Scripps Institute		Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.
WKPT-0293 Cl.2 rat kidney proximal tubule cell line			Made available by Dr. Ulrich Hopfer (Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH)