GFP-Expressing Cell Isolation and Analysis

Cells infected with the GFP virus were collected on 24 hpi using Hanks’-based, enzyme-free cell dissociation buffer (Gibco, 13150). Cells were washed once with PBS before they were fixed using 4% parafolmaldehyde (Electron Microscopy Sciences, USA) and suspended in fluorescence-activated cell sorting (FACS) buffer (PBS, 5% FBS). Cell suspensions were filtered through a 70-μm mesh, and resuspended in FACS buffer before analyzing them using an in-house flow cytometer (BD Accuri C6 Plus). A total of 2 × 10⁴ cells were collected for each sample, and the analysis was performed using FlowJo (version 10) (46 (link), 47 (link)).

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Yadavalli T., Suryawanshi R., Koganti R., Hopkins J., Ames J., Koujah L., Iqbal A., Madavaraju K., Agelidis A, & Shukla D. (2020). Standalone or combinatorial phenylbutyrate therapy shows excellent antiviral activity and mimics CREB3 silencing. Science Advances, 6(49), eabd9443.

Publication 2020

4% parafolmaldehyde BD Accuri C6 Plus

Buffer Cells Electron microscopy Enzyme Virus

Corresponding Organization : University of Illinois at Chicago

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Variable analysis

independent variables

Use of GFP virus to infect cells

dependent variables

Percentage of GFP-positive cells collected at 24 hours post-infection (hpi)

control variables

Cell dissociation using Hanks'-based, enzyme-free cell dissociation buffer
Washing cells with PBS before fixation
Fixation using 4% paraformaldehyde
Suspension of cells in FACS buffer (PBS, 5% FBS)
Filtering cell suspensions through a 70-μm mesh
Analysis using an in-house flow cytometer (BD Accuri C6 Plus)
Collection of 2 × 10^4 cells per sample
Analysis of data using FlowJo (version 10)

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