Endoplasmic Reticulum Calcium Measurements using Intracellular Fluorescent Dyes
Department
Biological Sciences
Major
Biology
Research Advisor
Aronstam, Robert
Advisor's Department
Biological Sciences
Funding Source
Missouri S&T cDNA Resource Center (www.cdna.org)
Abstract
Many cellular processes are regulated by changes in the concentration of calcium in the cytosol. The immediate source of cytosolic calcium is release from the endoplasmic reticulum (ER), while depletion of ER calcium leads to a secondary entry of calcium from the extracellular medium (this delayed influx is termed store-operated calcium entry; SOCE). We sought to validate an assay for ER calcium in CHO cells expressing human M1 muscarinic receptors. We loaded the cells with a Mag-fura-2 calcium sensitive dye. The large cytosolic signal was then depleted using a weak detergent (saponin). This revealed a large ER calcium concentration that could be quantified using a ratiometric single cell analysis. Our study found that pretreating the cells with 2.1 μM thapsigargin caused a 10-60% decrease in ER calcium content, further emphasizing that we successfully measured ER calcium with this method.
Biography
Corinna Edwards is a senior in Biological Sciences from Lebanon, TN. She has been doing research for Dr. Aronstam in the Neurobiology Lab since she transferred to Missouri S&T in the spring of 2014. She plans to graduate in December of 2015 and to pursue a master’s degree in nursing.
Research Category
Sciences
Presentation Type
Poster Presentation
Document Type
Poster
Location
Upper Atrium/Hall
Presentation Date
15 Apr 2015, 9:00 am - 11:45 am
Endoplasmic Reticulum Calcium Measurements using Intracellular Fluorescent Dyes
Upper Atrium/Hall
Many cellular processes are regulated by changes in the concentration of calcium in the cytosol. The immediate source of cytosolic calcium is release from the endoplasmic reticulum (ER), while depletion of ER calcium leads to a secondary entry of calcium from the extracellular medium (this delayed influx is termed store-operated calcium entry; SOCE). We sought to validate an assay for ER calcium in CHO cells expressing human M1 muscarinic receptors. We loaded the cells with a Mag-fura-2 calcium sensitive dye. The large cytosolic signal was then depleted using a weak detergent (saponin). This revealed a large ER calcium concentration that could be quantified using a ratiometric single cell analysis. Our study found that pretreating the cells with 2.1 μM thapsigargin caused a 10-60% decrease in ER calcium content, further emphasizing that we successfully measured ER calcium with this method.
