DS_Cell_Viability_ Kit- przeżywalność, metoda podwójnego barwienia

8/3/2019 DS_Cell_Viability_ Kit- przeżywalność, metoda podwójnego barwienia

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BD Biosciences

BD Biosciences2350 Qume DriveSan Jose, CA 95131-1807Tel 877.232.8995; Fax [email protected]

04/2007 23-6755-01

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

RESEARCHAPPLICATIONS

Studies of:• rapid counting of live/dead bacteria or other microbial cells

1-4

• efficacy of bacterial disinfectant

5

• viability of yeast during fermentation

6

• viability and concentration of cells in culture• viability and concentration of cells before staining for flow cytometric analysis• mammalian or microbial cells• viability and count of cells in bioreactors• viability of sperm for research studies

7,8

• viability in cell preparations containing debris

PRINCIPLES OF THEPROCEDURE

Flow cytometry provides a rapid and reliable method to quantify viable cells ineukaryotic 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 flowcytometry. The kit contains thiazole orange* (TO) solution to stain all cells andpropidium iodide (PI) to stain dead cells. BD™ Liquid Counting Beads is a liquidsuspension of fluorescent beads.† Add the beads to a flow sample to calculate absolutecounts. The kit can be ordered with or without counting beads. The method provides arapid alternative to manual microscopic methods.

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Live cells have intact membranes and are impermeable to dyes such as PI, which leaksinto cells with compromised membranes. TO is a permeant dye and enters all cells, liveand dead, to varying degrees. The fluorescent signal from TO in viable cells allows theirenumeration even when debris in the cell preparation contaminates a scatter gatearound the cells. Thus the combination of these two dyes provides a rapid and reliablemethod 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 µL 42 µmol/L TO in dimethyl sulfoxide (DMSO) and1 vial of 500 µL 4.3 mmol/L PI in water. The optional BD Liquid Counting Beads aresupplied as 1 vial of 10 mL of fluorescent microspheres in buffer with 0.1% sodiumazide.

Material Needed but notProvided

Recommended staining buffer: Physiologic phosphate-buffered saline containing 0.2%Pluronic® F-68 (BASF Corporation, Mount Olive, NJ, Catalog No. 51554728) and1 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 a0.22-µm filter.

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

BD Cell Viability Kit

Catalog No. 349483 100 Tests

Catalog No. 349480 with BD Liquid Counting Beads 100 Tests

Live and Dead CellDiscrimination



23-6755-01/page 2

HANDLING ANDSTORAGE

Store vials at 2° to 8°C 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.

WARNINGS

The toxicological properties of DMSO have not been determined. Keep container in awell-ventilated place (S9). Keep away from foodstuffs, beverages, and feed. Possiblerisks of irreversible effects (R40). Prolonged or repeated skin contact can causedermatitis. DMSO is readily absorbed through skin and can increase the tendency forother chemicals to penetrate the skin. Harmful by inhalation, in contact with skin, andif 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 contactwith eyes, rinse immediately with plenty of water and seek medical advice (S26). Thismaterial and its container must be disposed of as hazardous waste (S60). DMSO is notlisted (IARC, NTP, OSHA) as a cancer causing agent.

Sodium azide is present in this product. Sodium azide is harmful if swallowed. Keepaway from food, drink, and animal feedingstuff. Wear suitable protective clothing. If swallowed, seek medical advice immediately and show this container or label. Contactwith acids liberates very toxic gas. Azide compounds should be flushed with largevolumes of water during disposal to avoid deposits in lead or copper plumbing whereexplosive 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 approximateconcentration range of 5 x 10

5

to 10

7

cells/mL. (See Materials Needed but not Provided

for preparation method.) For other sample types, such as pharmaceutical, food orenvironmental samples, at least 100 organisms per mL need to be detected using flowcytometry. If necessary, samples can be brought into this range by an initialconcentration step.

Bacteria and yeast:

Add 5.0 µL of each dye solution to 500 µL of cell suspension. Thefinal staining concentrations are 420 nmol/L for TO and 43 µmol/L for PI. Vortex andincubate for at least 5 minutes at room temperature.

Mammalian cells:

Add 4.0 µL of TO and 2.0 µL of PI solution to 2 mL of cell

suspension. The final staining concentrations are 84 nmol/L for TO and 4.3 µmol/L forPI. Vortex and incubate for 5 minutes at room temperature.

Bull semen:

Add 2.0 µL of each dye solution to 2 mL of cell suspension. The finalstaining concentrations are 42 nmol/L for TO and 4.3 µmol/L for PI. Vortex andincubate for 5 minutes at room temperature.

NOTE

When using BD Liquid Counting Beads, allow the beads to come to roomtemperature. Prior to analysis, gently vortex the bead suspension for 30 seconds andadd 50 µL to each tube. Use the reverse pipetting technique to pipette for betteraccuracy. Cap the tubes and gently vortex to mix.

Acquisition and Analysis

Acquire prepared samples on a BD FACS™ brand flow cytometer equipped with488-nm laser excitation and BD CellQuest™ software. Use an FSC threshold formammalian cells (fluorescence threshold if also using BD Liquid Counting Beads), and

an SSC threshold for microbial cells. Gate cells using scatter and FL2.

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TO fluorescesprimarily in FL1 and FL2; PI fluoresces primarily in FL3. Therefore, the bestdiscrimination of live and dead populations is on an FL1 vs FL3 plot.



23-6755-01/page 3

To determine the concentration of the cell populations, use the following equation:

Representative Data

Figure 1 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a500-µL sample of E. coli

stained with 5 µL of TO and 5 µL of PI. Regions are setaround the live, injured, and dead bacterial populations; counting beads are not shown.

Figure 1

FL1 vs FL3 dot plot, gated on E. coli

by scatter

Figure 2 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a2-mL sample of PBMCs stained with 4 µL of TO and 2 µL of PI. Live cells are in R5;dead cells are in R3; and counting beads are in R4.

Figure 2

FL1 vs FL3 dot plot, gated on PBMCs by scatter

Figure 3 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a2-mL sample of Raji cells stained with 4 µL of TO and 2 µL of PI. Live cells are in R5;dead cells are in R3; and counting beads are in R4.

Figure 3

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

# events in cell region

# events in bead region-------------------------------------------------------

# beads/test*

test volume-------------------------------× dilution factor × concentration of cell population=

* This value is found on the vial of BD Liquid Counting Beads and can vary from lot to lot.

R6

R5

R4

live

injured

dead

FL1-H

F L 3 - H

R3

R4

R5live

dead

injured

FL1-H

F L 3 - H

beads

FL1-H

F L 3 - H

R3

R4

R5

dead

injured

live

beads



23-6755-01/page 4

Figure 4 shows results obtained by staining a 2-mL sample of thawed bull semen with2 µL of TO and 2 µL of PI. For staining, 20 µL of thawed semen was added to 2 mL of staining buffer. Live sperm are in R3, dead sperm are in R2, and injured sperm arebetween the two regions.

Figure 4

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

LIMITATIONS

Dispose of stained samples, extra dye solution, and container according to local, state,and federal regulations.

CHARACTERIZATION

To ensure consistently high-quality reagents, each lot of reagent is tested forconformance with characteristics of a standard reagent. Representative flow cytometricdata is included in this data sheet.

WARRANTY

Unless otherwise indicated in any applicable BD general conditions of sale for non-UScustomers, 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. BD’S 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.

REFERENCES

1. Nebe-von-Caron G, Stephens PJ, Badley AR. Bacterial detection and differentiation by cytometry andfluorescent 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 fluorescence flow 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 flow 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 efficacy using flow

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 flow cytometry. Microbial Cytometry Application Note.

BD Biosciences,

Immunocytometry Systems; San Jose, CA. 2002.

7. Graham JK. Assessment of sperm quality: a flow 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 flowcytometry: simultaneous analysis of sperm count and sperm viability. J Toxicol Sci

. 1998;23:373-378.

FL1-H

F L 3 - H

live

injured

dead

R2

R3

DS_Cell_Viability_ Kit- przeżywalność, metoda podwójnego barwienia

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