Live and Dead Cell Discrimination

BD Cell Viability Kit

Catalog No. 349483 Catalog No. 349480 with BD Liquid Counting Beads 100 Tests 100 Tests

RESEARCH APPLICATIONS

Studies of: rapid counting of live/dead bacteria or other microbial cells1-4 efcacy of bacterial disinfectant5 viability of yeast during fermentation6 viability and concentration of cells in culture viability and concentration of cells before staining for ow cytometric analysis mammalian or microbial cells viability and count of cells in bioreactors viability of sperm for research studies7,8 viability in cell preparations containing debris

PRINCIPLES OF THE PROCEDURE

Flow cytometry provides a rapid and reliable method to quantify viable cells in eukaryotic and prokaryotic cell suspensions.1-3,7,8 The BD Cell Viability Kit offers an easy-to-use dye combination to distinguish live and dead cells for analysis by ow cytometry. The kit contains thiazole orange* (TO) solution to stain all cells and propidium iodide (PI) to stain dead cells. BD Liquid Counting Beads is a liquid suspension of uorescent beads. Add the beads to a ow sample to calculate absolute counts. The kit can be ordered with or without counting beads. The method provides a rapid alternative to manual microscopic methods.4-6 Live cells have intact membranes and are impermeable to dyes such as PI, which leaks into cells with compromised membranes. TO is a permeant dye and enters all cells, live and dead, to varying degrees. The uorescent signal from TO in viable cells allows their enumeration even when debris in the cell preparation contaminates a scatter gate around the cells. Thus the combination of these two dyes provides a rapid and reliable method for discriminating live and dead eukaryotic and prokaryotic cells, including peripheral blood mononuclear cells (PBMCs), mammalian cell lines, bacteria, and yeast.

MATERIALS PROVIDED

The kit contains 1 vial of 500 L 42 mol/L TO in dimethyl sulfoxide (DMSO) and 1 vial of 500 L 4.3 mmol/L PI in water. The optional BD Liquid Counting Beads are supplied as 1 vial of 10 mL of uorescent microspheres in buffer with 0.1% sodium azide. Recommended staining buffer: Physiologic phosphate-buffered saline containing 0.2% Pluronic F-68 (BASF Corporation, Mount Olive, NJ, Catalog No. 51554728) and 1 mmol/L EDTA (Sigma, St. Louis, MO, Catalog No. ED2SS). NOTE For bacteria, 0.01% Tween-20 (Sigma, St. Louis, MO, Catalog No. P-1379) can be substituted for the Pluronic F-68. The staining buffer should be passed through a 0.22-m lter.

Material Needed but not Provided

* US Patent Nos. 4,883,867; 4,957,870 US Patent Nos. 5,723,218; 5,187,288 (Molecular Probes)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 Tel 877.232.8995; Fax 800.325.9637 facservice@bd.com 04/2007 23-6755-01

BD Biosciences

HANDLING AND STORAGE WARNINGS

Store vials at 2 to 8C with the TO and PI stored in the desiccated container provided. Each reagent is stable for the period shown on the bottle label when stored as directed. The toxicological properties of DMSO have not been determined. Keep container in a well-ventilated place (S9). Keep away from foodstuffs, beverages, and feed. Possible risks of irreversible effects (R40). Prolonged or repeated skin contact can cause dermatitis. DMSO is readily absorbed through skin and can increase the tendency for other chemicals to penetrate the skin. Harmful by inhalation, in contact with skin, and if swallowed (R20/21/22). After contact with skin, wash immediately with plenty of water (S28). Wear suitable protective clothing and gloves (S36/37). After swallowing, if symptoms persist, consult a doctor. Avoid contact with eyes (S25). In case of contact with eyes, rinse immediately with plenty of water and seek medical advice (S26). This material and its container must be disposed of as hazardous waste (S60). DMSO is not listed (IARC, NTP, OSHA) as a cancer causing agent. Sodium azide is present in this product. Sodium azide is harmful if swallowed. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing. If swallowed, seek medical advice immediately and show this container or label. Contact with acids liberates very toxic gas. Azide compounds should be ushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop. Thiazole orange and propidium iodide are possible mutagens.

METHOD

Recommended amounts of TO and PI depend on the cell type being stained. Dilute cultured cells or PBMCs at least 1:10 in staining buffer to an approximate concentration range of 5 x 105 to 107 cells/mL. (See Materials Needed but not Provided for preparation method.) For other sample types, such as pharmaceutical, food or environmental samples, at least 100 organisms per mL need to be detected using ow cytometry. If necessary, samples can be brought into this range by an initial concentration step. Bacteria and yeast: Add 5.0 L of each dye solution to 500 L of cell suspension. The nal staining concentrations are 420 nmol/L for TO and 43 mol/L for PI. Vortex and incubate for at least 5 minutes at room temperature. Mammalian cells: Add 4.0 L of TO and 2.0 L of PI solution to 2 mL of cell suspension. The nal staining concentrations are 84 nmol/L for TO and 4.3 mol/L for PI. Vortex and incubate for 5 minutes at room temperature. Bull semen: Add 2.0 L of each dye solution to 2 mL of cell suspension. The nal staining concentrations are 42 nmol/L for TO and 4.3 mol/L for PI. Vortex and incubate for 5 minutes at room temperature. NOTE When using BD Liquid Counting Beads, allow the beads to come to room temperature. Prior to analysis, gently vortex the bead suspension for 30 seconds and add 50 L to each tube. Use the reverse pipetting technique to pipette for better accuracy. Cap the tubes and gently vortex to mix.

Acquisition and Analysis

Acquire prepared samples on a BD FACS brand ow cytometer equipped with 488-nm laser excitation and BD CellQuest software. Use an FSC threshold for mammalian cells (uorescence threshold if also using BD Liquid Counting Beads), and an SSC threshold for microbial cells. Gate cells using scatter and FL2.4-6 TO uoresces primarily in FL1 and FL2; PI uoresces primarily in FL3. Therefore, the best discrimination of live and dead populations is on an FL1 vs FL3 plot.

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To determine the concentration of the cell populations, use the following equation:
# events in cell region # beads/test* ------------------------------------------------------ ------------------------------ dilution factor = concentration of cell population # events in bead region test volume
* This value is found on the vial of BD Liquid Counting Beads and can vary from lot to lot.

Representative Data

Figure 1 shows results obtained by adding 50 L of BD Liquid Counting Beads to a 500-L sample of E. coli stained with 5 L of TO and 5 L of PI. Regions are set around the live, injured, and dead bacterial populations; counting beads are not shown.
R6 dead

R5 FL3-H injured

R4

live

FL1-H

Figure 1 FL1 vs FL3 dot plot, gated on E. coli by scatter

Figure 2 shows results obtained by adding 50 L of BD Liquid Counting Beads to a 2-mL sample of PBMCs stained with 4 L of TO and 2 L of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.
dead R3

beads FL3-H R4

injured

R5 live

FL1-H

Figure 2 FL1 vs FL3 dot plot, gated on PBMCs by scatter

Figure 3 shows results obtained by adding 50 L of BD Liquid Counting Beads to a 2-mL sample of Raji cells stained with 4 L of TO and 2 L of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.
R3 dead

beads FL3-H R4 injured

live

R5

FL1-H

Figure 3 FL1 vs FL3 dot plot, gated on Raji cells by scatter

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Figure 4 shows results obtained by staining a 2-mL sample of thawed bull semen with 2 L of TO and 2 L of PI. For staining, 20 L of thawed semen was added to 2 mL of staining buffer. Live sperm are in R3, dead sperm are in R2, and injured sperm are between the two regions.
R2

dead

FL3-H

injured

R3 live

FL1-H

Figure 4 FL1 vs FL3 dot plot, gated on thawed bull semen by scatter

LIMITATIONS CHARACTERIZATION

Dispose of stained samples, extra dye solution, and container according to local, state, and federal regulations. To ensure consistently high-quality reagents, each lot of reagent is tested for conformance with characteristics of a standard reagent. Representative ow cytometric data is included in this data sheet. Unless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE. BDS SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

WARRANTY

REFERENCES

1. Nebe-von-Caron G, Stephens PJ, Badley AR. Bacterial detection and differentiation by cytometry and uorescent probes. Proc Royal Microbiol Society. 1999;34:321-327. 2. Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multi-colour uorescence ow cytometry and single cell sorting. J Microbiol Meth. 2000;42:97-114. 3. Davey HM, Kell DB. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev. 1996;60:641-696. 4. Alsharif R, Godfrey W. Bacterial detection and live/dead discrimination by ow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002. 5. Alsharif R, Tapia M, Godfrey W, Wanalund J, Nagar M. Bacterial disinfectant efcacy using ow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002. 6. Thornton R, Godfrey W, Gilmour L, Alsharif R. Evaluation of yeast viability and concentration during wine fermentation using ow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002. 7. Graham JK. Assessment of sperm quality: a ow cytometric approach. Anim Reprod Sci. 2001;68:239-247. 8. Yamamoto T, Mori S, Yoneyama M, Imanishi M, Takeuchi M. Evaluation of rat sperm by ow cytometry: simultaneous analysis of sperm count and sperm viability. J Toxicol Sci. 1998;23:373-378.

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