Title

Endoplasmic Reticulum Calcium Measurements using Intracellular Fluorescent Dyes

Presenter Information

Corinna Edwards

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

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Apr 15th, 9:00 AM Apr 15th, 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.