Viability Staining

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Flow Cytometry BestProtocols Page 1 of 3

Viability Staining

Research Use Only

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 [email protected]

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 theeBioscience Technical Support websitefor 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. Uponentering dead cells, Propidium Iodide will intercalate into double-

stranded DNA or double-stranded RNA in a stoichiometric manner, while 7-AAD will intercalate only with double-

stranded 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. After staining cells for surface antigens, wash cells 1-2 times with flow cytometry staining buffer2. Resuspend cells in an appropriate volume of flow cytometry staining buffer3. For every 100 L of cells, add 5 L of Propidium Iodide or 7-AAD Staining Solution4. 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 seeStaining Dead Cells with eBioscienceFixable Viability Dyes

for staining dead cells with viability dyes that are compatible with intracellular staining protocols.
http://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/technical-support.htmhttp://www.ebioscience.com/propidium-iodide-staining-solution.htmhttp://www.ebioscience.com/propidium-iodide-staining-solution.htmhttp://www.ebioscience.com/propidium-iodide-staining-solution.htmhttp://www.ebioscience.com/7-aad-viability-staining-solution.htmhttp://www.ebioscience.com/7-aad-viability-staining-solution.htmhttp://www.ebioscience.com/7-aad-viability-staining-solution.htmhttp://www.ebioscience.com/7-aad-viability-staining-solution.htmhttp://www.ebioscience.com/propidium-iodide-staining-solution.htmhttp://www.ebioscience.com/technical-support.htm


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Viability Staining

Research Use Only

Revised 10-5-2010



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)


Calcein Dyes are lyophilized and should be reconstituted in anhydrous DMSO before use. Reconstituted dye should

be used within a short period of timeafter reconstitution. For short-term storage, store at -20C with dessicant.

Avoid freeze-thawingand allow the vial to equilibrate to room temperature before opening.

1. Prepare cells as desired2. Resuspend 1-5x106cells in an appropriate volume of flow cytometry staining buffer(0.1 1 mL)3. 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 PBS4. Incubate at room temperature for 30 minutes.5. Wash cells 1-2 times with flow cytometry staining buffer6. 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.
http://www.ebioscience.com/calcein-am-viability-dye-ultrapure-grade.htmhttp://www.ebioscience.com/calcein-am-viability-dye-ultrapure-grade.htmhttp://www.ebioscience.com/calcein-am-viability-dye-ultrapure-grade.htmhttp://www.ebioscience.com/calcein-violet-450-am-viability-dye.htmhttp://www.ebioscience.com/calcein-violet-450-am-viability-dye.htmhttp://www.ebioscience.com/calcein-violet-450-am-viability-dye.htmhttp://www.ebioscience.com/calcein-blue-am-viability-dye.htmhttp://www.ebioscience.com/calcein-blue-am-viability-dye.htmhttp://www.ebioscience.com/calcein-blue-am-viability-dye.htmhttp://www.ebioscience.com/calcein-blue-am-viability-dye.htmhttp://www.ebioscience.com/calcein-violet-450-am-viability-dye.htmhttp://www.ebioscience.com/calcein-am-viability-dye-ultrapure-grade.htm


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Viability Staining

Research Use Only

Revised 10-5-2010



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)


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. Prepare cells as desired.2. Wash cells 2 times in azide-free and serum/protein-free PBS.3. Resuspend cells at 1-10x106/mL in azide-free and serum/protein-free PBS.4. Add 1 L of Fixable Viability Dye per 1 mL of cells and vortex immediately.5. Incubate for 30 minutes at 2-8C, protect from light.6. Wash cells 1-2 times with flow stain buffer or equivalent.7. 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.
http://www.ebioscience.com/fixable-viability-dye-efluor-450.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-450.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-450.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-660.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-660.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-660.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-780.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-780.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-780.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-780.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-660.htmhttp://www.ebioscience.com/fixable-viability-dye-efluor-450.htm

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