Cell Cycle Staining With PI for GFP Transfected Cells Using PFA_EtOH

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Analytical Cytometry/ Image Analysis Core Facility; EOHSI; (732) 445-0211 Cell Cycle Staining with PI for Cytoplasmic GFP transfected Cells using PFA and Ethanol Theresa Choi Date: 3-4-2010 page 1 of 2 Cell cycle staining with Propidium Iodide for the cytoplasmic GFP transfected cells using Paraformaldehyde and Ethanol 1. Test principle For optimal PI staining, cells require permeabilization with ethanol to allow the dye access to the nucleus. However, such treatment with ethanol results in
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  Analytical Cytometry/ Image Analysis Core Facility; EOHSI; (732) 445-0211 Theresa ChoiCell Cycle Staining with PI for Cytoplasmic GFP transfected Cells usingPFA and Ethanol Date: 3-4-2010 page 1 of 2 Cell cycle staining with Propidium Iodide for the cytoplasmic GFP transfectedcells using Paraformaldehyde and Ethanol   1. Test principle For optimal PI staining, cells require permeabilization with ethanol to allow the dye access to thenucleus. However, such treatment with ethanol results in the loss of GFP fluorescence as thesoluble, cytoplasmic GFP leaks out of the cells following permeabilization. In contrast, the use offixatives (e.g. paraformaldehyde) that retain GFP in the cells leads to DNA histograms withunacceptably high codfficients of variations (CV) for the G1/G0 peaks.A membrane-localized or a transmembrane GFP fusion protein retained in cells following ethanolpermeabilization facilitating the simultaneous detection of GFP (transfection marker) and high-resolution PI data (cell cycle analysis).This protocol is for cell fixation/ permeabilization for combined measurement of GFP expressionand PI DNA content. Cells are fixed with 1% formaldehyde followed by treatment with 70%ethanol for cell membrane solubilization, and then stained with PI in the presence of RNAse A forDNA content. 2. Specimen GFP transfected cells 3. Materials and reagents ã 5ml Falcon polypropylene Round-Bottom test tubes (12x75 mm) ã cooling centrifuge ã 37 ° C water bath ã 40 μ m nylon mesh (BD Falcon Cell Strainer # 352340) or 70 μ m nylon mesh ã sterile-filtered Phosphate-buffered saline (PBS), 4 ° C ã   Fixation Solution (4 ° C): Add 2g high purity paraformaldehyde (electron microscopygrade from Polysciences, 2% w/v final) to 100 ml PBS. Heat to 70 ° C in a fume hood untilthe paraformaldehyde completely dissolves (~1hr). Cool to room temperature and adjustpH to 7.2. Store at 2 ° C to 8 ° C protected from light. The solution is stable for at least 1month. Check pH periodically. ã   70% ethanol , -20 ° C ã   Propidium iodide (PI) solution : Stock solution: Dissolve 1 mg PI (Sigma, P 4170) in 1 ml dH 2 O at 1 mg/ml.Store aliquots at -20 ° C for up to 2 years. Aliquots that are frequently used can be storedat 4 ° C protected from light for up to 2 months. Discard solutions of PI that have beenexposed to room temperature for more than 48 hr, or if they appear dark red. Keep it inthe dark. Working solution: Dilute PI stock solution 1:25 with PBS. Final concentration of PI is 40 μ g/ml. ã   RNAse A solution:  Dissolve 50 mg RNAse A (Sigma, R-4875) in 50 ml of PBS and adjust to 0.1% Tween-20.Place solution into a 95 ° C water bath for 30 min.Allow the solution to cool on ice for 1 hr. Remove precipitate by filtering through a 0.2 μ mfilter.Solution can be stored for up to 6 months at 4 ° C or in aliquots be frozen at -20 ° C. 4. Controls ã Cells transfected with GFP alone without PI added ã No GFP transfected and no drug treated cells stained with PI  Analytical Cytometry/ Image Analysis Core Facility; EOHSI; (732) 445-0211 Theresa ChoiCell Cycle Staining with PI for Cytoplasmic GFP transfected Cells usingPFA and Ethanol Date: 3-4-2010 page 2 of 2 5. Procedure a) Count cellsb) Place 1x 10 6 cells in to a 12x75 mm test tube and wash once with PBS by centrifuging 5min at 300 x g, 4 ° C.c) Fix cells with paraformaldehyde: Remove supernatant by aspiration and add 500 μ l coldPBS to the cell pellet. Mix gently. Add 500 μ l cold (4 ° C) fixation solution and mix again.Incubate 1 hr at 4 ° C.    Different concetrations of formadldehyde fixative may be needed for optimal retention of GFP in various cell types, and for obtaining acceptable CV on DNAhistograms. d) Permeabilize cells with ethanol: Centrifuge cells 5 min at 300 x g, 4 ° C. Remove supernatantby aspiration and wash once with cold 3 ml PBS. Add 1 ml 70% ethanol at -20 ° C drop-wiseto the cell pellet with the tube sitting on a vortex mixer. Incubate cell suspension overnightat 4 ° C.    Be careful for aspiration of the supernatant since a cell pellet may not be visible after the fixation steip with formaldehyde. Fixed cells aggregate less well and tend to spread out at the bottom of the tube.    Vortexing cells gently during the addition of ethanol can reduce the formation of cell clumps, but too vigorous vortexing can lead to cell disruption.    Incubation times with 70% ethanol should not be less than 2 hrs. e) Stain cells with PI: Centrifuge cells 5 min at 300 x g, 4 ° C. Remove supernatant and add 1mlpropidium iodide (PI) working solution. Incubate cell suspension 30 min in a 37 ° C waterbath in the dark. If needed, filter samples through a 40 or 70 um nylon mesh to removeclumps.f) Analyze samples on the flow cytometer. Note: PI is a nucleic acid-specific, red-fluorescent dye. It is also a suspected carcinogen, soemploy appropriate safety precautions when working with this dye. Reference: Current Protocols in Cytometry (2001) 7.16.1, Ingrid Schmid and Kathleen M. Sakamoto
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