Trypan Blue and Ischemia: A measure of cell viability
Mentor 1
James Moyer
Location
Union Wisconsin Room
Start Date
27-4-2018 1:00 PM
Description
According to the National Institutes for Neurological Disorders and Stroke (NINDS), ischemic strokes account for 87% of all strokes. Ischemic strokes result when blood flow to a region of the brain is disrupted, which thus deprives neurons within the affected brain regions of valuable oxygen and nutrients. To study the way in which strokes affect the brain, various cell culture, in vivo, and in vitro methods have been developed. Such models enable scientists to evaluate the cellular mechanisms that contribute to ischemic cell death as well as explore the efficacy of various treatments. Our lab uses an in vitro technique in which brain slices from a rat undergo oxygen and glucose deprivation or OGD. This is done by briefly replacing the normal oxygenated artificial cerebrospinal fluid (aCSF; bubbled with 95% O2/5% CO2) with, glucose-free aCSF (bubbled with 95% N2/5% CO2). After 5-min of OGD, reperfusion is accomplished by placing slices back into an oxygenated aCSF solution, which mimics (in a controlled setting) the basic events associated with an ischemic stroke. Slices are then subsequently fixed with formalin. In order to quantify cell death associated with OGD, trypan blue is included in the aCSF during reperfusion. Trypan Blue is not able to pass through the semi-permeable membrane of living cells and consequently is only taken up by dead or dying cells whose cell membrane has begun to break down and become leaky. Thus, it is possible to assess cell death by counting dead or dying cells that are stained with Trypan Blue. This technique is often referred to the “Trypan Blue exclusion” test for cell viability because healthy, viable cells remain unstained. This preparation allows us to study possible therapeutic agents that could have neuroprotective effects on ischemic strokes.
Trypan Blue and Ischemia: A measure of cell viability
Union Wisconsin Room
According to the National Institutes for Neurological Disorders and Stroke (NINDS), ischemic strokes account for 87% of all strokes. Ischemic strokes result when blood flow to a region of the brain is disrupted, which thus deprives neurons within the affected brain regions of valuable oxygen and nutrients. To study the way in which strokes affect the brain, various cell culture, in vivo, and in vitro methods have been developed. Such models enable scientists to evaluate the cellular mechanisms that contribute to ischemic cell death as well as explore the efficacy of various treatments. Our lab uses an in vitro technique in which brain slices from a rat undergo oxygen and glucose deprivation or OGD. This is done by briefly replacing the normal oxygenated artificial cerebrospinal fluid (aCSF; bubbled with 95% O2/5% CO2) with, glucose-free aCSF (bubbled with 95% N2/5% CO2). After 5-min of OGD, reperfusion is accomplished by placing slices back into an oxygenated aCSF solution, which mimics (in a controlled setting) the basic events associated with an ischemic stroke. Slices are then subsequently fixed with formalin. In order to quantify cell death associated with OGD, trypan blue is included in the aCSF during reperfusion. Trypan Blue is not able to pass through the semi-permeable membrane of living cells and consequently is only taken up by dead or dying cells whose cell membrane has begun to break down and become leaky. Thus, it is possible to assess cell death by counting dead or dying cells that are stained with Trypan Blue. This technique is often referred to the “Trypan Blue exclusion” test for cell viability because healthy, viable cells remain unstained. This preparation allows us to study possible therapeutic agents that could have neuroprotective effects on ischemic strokes.
