Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

Summary

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

Abstract

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H₂DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H₂DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H₂DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H₂DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H₂DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

Protocol

1. Cell culture of feline astrocytes and treatment with H₂O₂

1. Culture cell line G355-5 in a 75 cm² flask with DMEM media plus nutrients at 37°C and 5% CO₂ until 60% confluent (˜ 5 days). Replace the media every 1-2 days or as needed to maintain a healthy culture.
2. Remove media and add 2 ml of 0.25% trypsin. Lift the cells by pipetting for 45 s - 1 min at room temperature (RT).
3. Quickly transfer the suspension to a 15 ml conical tube containing 10 ml of media. Mix gently.
4. Centrifuge at 300 x g, 23°C for 3 min to obtain a pellet. Discard the supernatant without disturbing the pellet.
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Discussion

An often suggested mechanism of virus induced neuronal damage is oxidative stress ^{1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31}. Basically, it is proposed that through viral exposure the glia (astrocytes and microglia) release reactive oxygen radicals such as, hydroxide, superoxide anion, nitric oxide, and/or hydrogen peroxide, which are toxic to neurons. Similarly, oxidative stress has also been suggested as a major pathologic mechanism in methamphetamine-induced neurotoxicity ^{2, 6, 17}. Since our group i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Cellgro	15-013-CV
MEM Vitamins	Cellgro	25-020-cl	1% (v/v)
L-glutamine	Hyclone	17-605E	1% (v/v)
Non Essential Amino Acids	Hyclone	25-025-cl	1% (v/v)
FBS	Hyclone	SH30071.03	20% (v/v)
Essential Amino Acids	Cellgro	25-030-cl	0.2% (v/v)
500ml vacuum filtration system	VWR international	87006-076
15 ml conical tubes	Falcon BD	352097
75 cm2 tissue culture flasks	Falcon BD	353136
6 well tissue culture plate	Falcon BD	353224
CM-H2DCFDA	Invitrogen	C6827
Propidium Iodide (PI)	Sigma-Aldrich	P4170-10mg
Trypsin	Lonza Inc.	17-160F
H2O2	Sigma-Aldrich	H6520
HyQ Antibiotic	Hyclone	SV30079.01	0.1% (v/v)
G355-5 cells	ATCC	CRL-2033	Normal feline brain