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Cell Cycle Analysis Using Propidium Iodide Staining with GFP Detection
Published: Apr 5, 2012 DOI: 10.21769/BioProtoc.199 Views: 24432
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Abstract
Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.
Keywords: Cell cycle
GFP
FACS
Propidium iodide
Background
Materials and Reagents
- Phosphate buffered saline (PBS)
- Glucose (Sigma-Aldrich, catalog number: G8270 )
- Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15170 )
- 70% EtOH
- Hepes (Sigma-Aldrich, catalog number: H3375 )
- NP-40 (MBL International, catalog number: JM-2111-100 )
- BSA (Sigma-Aldrich, catalog number: A3803 )
- RNase A (Sigma-Aldrich, catalog number: R4642 )
- Fix solution (see Recipes)
- Wash solution (see Recipes)
Equipment
- Centrifuges (Beckman Falcon, TLS-55 )
- 15 ml polypropylene falcon tubes (BD Biosciences, Falcon®, catalog number: 352097 )
- FACS machine
Procedure
Category
Cell Biology > Cell imaging > Fluorescence
Biochemistry > Protein > Fluorescence
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