Flow Cytometry BestProtocols Page 1 of 3

Viability Staining
Research Use Only
Protocol A: Staining Dead Cells with Propidium Iodide or 7-AAD Protocol B: Staining Live Cells with Calcein Dyes Protocol C: Staining Dead Cells with eBioscience Fixable Viability Dyes

Introduction
Viability staining is an essential component of any flow cytometry experiment. Dead cells can compromise the integrity of the data by non-specifically binding antibodies; therefore it is essential that dead cells be excluded from analysis. eBiosicence offers several different dyes that can be used to discriminate live and dead cells. Please visit the eBioscience Technical Support website for more information to help determine which dyes are suitable for the specific application of interest.

Protocol A: Staining Dead Cells with Propidium Iodide or 7-AAD

Propidium Iodide and 7-AAD can be used to stain dead cells so that they may be excluded from analysis in standard cell surface staining protocols. These dyes cannot pass through intact cell membranes, but may freely enter cells with compromised cell membranes. Upon entering dead cells, Propidium Iodide will intercalate into doublestranded DNA or double-stranded RNA in a stoichiometric manner, while 7-AAD will intercalate only with doublestranded DNA. Because this intercalation is mediated by non-covalent forces, these dyes must remain present in the buffer used to resuspend cells for data acquisition so that dead cells will remain labeled. Reagents Propidium Iodide Staining Solution (cat. 00-6990) 7-AAD Viability Staining Solution (cat. 00-6993)

Experimental Procedure
1. 2. 3. 4. After staining cells for surface antigens, wash cells 1-2 times with flow cytometry staining buffer Resuspend cells in an appropriate volume of flow cytometry staining buffer For every 100 L of cells, add 5 L of Propidium Iodide or 7-AAD Staining Solution Incubate for 5 15 minutes on ice or at room temperature before analyzing cells on a flow cytometer Note: Propidium Iodide and 7-AAD must remain in the buffer during acquisition. Do not wash cells after the addition of Propidium Iodide or 7-AAD. Cells should be analyzed as soon as possible after the initial incubation period due to adverse effects on the viability of cells left in the presence of propidium iodide or 7-AAD for too long.

Note: Neither Propidium Iodide nor 7-AAD can be used to discriminate live and dead cells when intracellular staining is desired. Please see Staining Dead Cells with eBioscienceFixable Viability Dyes for staining dead cells with viability dyes that are compatible with intracellular staining protocols.

Revised 10-5-2010 Provided as a courtesy by eBioscience, Inc. Copyright 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 Fax: 858.642.2046 www.ebioscience.com info@ebioscience.com

Flow Cytometry BestProtocols Page 2 of 3

Viability Staining
Research Use Only

Protocol B: Staining Live Cells with Calcein Dyes

Calcein AM, Calcein Violet AM, and Calcein Blue AM are non-fluorescent, membrane-permeable dyes that can be used to identify and label live cells. Upon entering the cells, the dyes are converted to membrane-impermeable, fluorescent compound by intracellular esterases. Dead cells will not have active esterase activity nor will they retain the fluorescent dyes. Thus, the calcein dyes allow viable cells to be fluorescently labeled for analysis in standard cell surface staining protocols. Reagents Calcein AM (cat. 65-0853) Calcein Violet AM (cat. 65-0854) Calcein Blue AM (cat. 65-0855)

Experimental Procedure
Calcein Dyes are lyophilized and should be reconstituted in anhydrous DMSO before use. Reconstituted dye should be used within a short period of time after reconstitution. For short-term storage, store at -20C with dessicant. Avoid freeze-thawing and allow the vial to equilibrate to room temperature before opening. 1. 2. 3. Prepare cells as desired 6 Resuspend 1-5x10 cells in an appropriate volume of flow cytometry staining buffer (0.1 1 mL) Add calcein dye at the desired concentration and mix well (please see technical data sheet for the specific calcein dye of interest for a recommended concentration range) Note: It may be necessary to make a more dilute working solution of the dye, which may be done with flow cytometry staining buffer or PBS 4. Incubate at room temperature for 30 minutes. 5. Wash cells 1-2 times with flow cytometry staining buffer 6. Proceed with surface staining or analysis on a flow cytometer, as desired.

Note: Staining with the calcein dyes may be done before or after surface staining with antibodies. It is recommended that the dyes be titrated by each investigator for optimal performance in the assay of interest. Because the calcein dyes are not retained in cells with compromised cell membranes they are not compatible with intracellular staining protocols. Please see Staining Dead Cells with Fixable Viability Dyes for staining dead cells with viability dyes that are compatible with intracellular staining protocols.

Revised 10-5-2010 Provided as a courtesy by eBioscience, Inc. Copyright 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 Fax: 858.642.2046 www.ebioscience.com info@ebioscience.com

Flow Cytometry BestProtocols Page 3 of 3

Viability Staining
Research Use Only

Protocol C: Staining Dead Cells with eBioscience Fixable Viability Dyes

The eBioscience Fixable Viability Dyes can be used to irreversibly label dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells labeled with Fixable Viability Dyes can be washed, fixed, permabilized, and stained for intracellular antigens without any loss of staining intensity of the dead cells. Thus, Fixable Viability Dyes are fully compatible with intracellular staining protocols.

Reagents Fixable Viability Dye eFluor 450 (cat. 65-0863) Fixable Viability Dye eFluor 660 (cat. 65-0864) Fixable Viability Dye eFluor 780 (cat. 65-0865)

Experimental Procedure
Allow vial of Fixable Viability Dye to equilibrate to room temperature before opening. Staining with Fixable Viability Dye must be done in azide-free and serum/protein-free PBS. For consistent staining of cells, do not stain cells in less than 0.5 ml. 1. 2. 3. 4. 5. 6. 7. Prepare cells as desired. Wash cells 2 times in azide-free and serum/protein-free PBS. 6 Resuspend cells at 1-10x10 /mL in azide-free and serum/protein-free PBS. Add 1 L of Fixable Viability Dye per 1 mL of cells and vortex immediately. Incubate for 30 minutes at 2-8C, protect from light. Wash cells 1-2 times with flow stain buffer or equivalent. Fix and/or permeabilize cells as desired.

Note: Cells may be stained with Fixable Viability Dyes before or after surface staining. After staining with Fixable Viability Dyes, cells may also be cyropreserved for analysis at a later time. It is recommended that each investigator determine the optimal concentration for the assay of interest.

Revised 10-5-2010 Provided as a courtesy by eBioscience, Inc. Copyright 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 Fax: 858.642.2046 www.ebioscience.com info@ebioscience.com