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DAPIprotocol

For life science research only. Not for use in diagnostic procedures.

FOR IN VITRO USE ONLY.

DAPI (4’,6-Diamidine-2’-phenylindole dihydrochloride)

Powder, non sterile

Cat. No. 10 236 276 001Version January 2007 10 mg Store this kit at +15 to +25°C

1.Product overview

Formulation Powder (crystallized); non sterile

Properties

Typical analysis?90% (from N.)

Application Detection of mycoplasmal infections of cell cultures.

Assay principle The fluorescent dye DAPI binds selectively to DNA and

forms strongly fluorescent DNA-DAPI complexes with

high specificity.

On adding DAPI to tissue culture cells it is rapidly

taken up into cellular DNA yielding highly fluorescent

nuclei and no detectable cytoplasmic fluorescence. If

the cells are contaminated with mycoplasmas, charac-

teristic discrete fluorescent foci are readily detected

over the cytoplasm and sometimes in intercellular

spaces.

Reconstitution In 2–10 ml double dist. water; 1–5 mg/ml final concen-

tration.

Note: Prepare aliquots and store at ?15 to ?25°C.

Storage stability Stable at +15 to +25°C, protected from light, until the

expiration date printed on the label.

Background information Figures on the incidence of mycoplasmal infections of

cell cultures range from 1–92% (1-4). The origins of

mycoplasmal infection of cell cultures are bovine

serum (A. laidlawii, M. arginini, M. hyorhinis), labora-

tory personnel (M. orale) and mycoplasma-infected

cultures.

Mycoplasmas produce various effects on the infected

cell culture (2–4). Mycoplasmal infection cannot be

detected by naked eye other than by signs of detoria-

tion in the culture. It is important to appreciate that

mycoplasmas do not always reveal their presence with

macroscopic alterations of the cells or media. Many

mycoplasma contaminants, particularly in continuous

cell lines grow slowly and do not destroy host cells.

Therefore there is an absolute requirement for routine,

periodic assays for possible covert contamination of all

cell cultures, particularly continuous or established cell

lines.

Formula C

16

H

15

N

5

× 2 HCl

Molecular weight M

r

350.3

Solubility in water25 mg/ml

Absorbance maximum in

aqueous solution

? ? 340 nm

Emission maximum in

aqueous solution

? ? 488 nm

Solution Storage/stability

Stock solution (1–5 mg/ml)?15 to ?25°C for

12months

Working solution (1?g/ml)+2 to +8°C for about 6

months

Detection

techniques

A variety of techniques have been developed for the

detection of cell culture mycoplasmas, e.g.

?DNA staining,

?mycoplasma-mediated cytotoxicity,

?biochemical detection methods,

?electron microscopy,

?ELISA (Mycoplasma Detection Kit*) or by PCR

(Mycoplasma PCR ELISA*) (2, 3, 5).

DNA staining employing fluorescent dyes that bind

specifically to DNA is the most popular method. This

method is quick and simple to perform. Two dyes, 4’,6-

diamidine-2’-phenylindole (DAPI) and bisbenzimide

(H33258) have been widely used (6–8). The rationale

behind this assay is that mycoplasma-free cultures

exhibit only nuclear fluorescence. Mycoplasma-

infected cultures also display extranuclear fluores-

cence. Mitochondrial DNA is not apparent in prepara-

tions stained either with DAPI or H33258.

2. Procedures and required materials

2.1 Before you begin

General

considerations

Prior to the assay cell cultures should be passed in

antibiotic-free media for a minimum of two passages.

The cultures should be assayed 3–4 days after pas-

sage. The cell supernatant will contain 107–108 CFU/ml,

additional organism are adsorbed onto host cells.

Preparation of

stock solution

Dissolve in double dist. water to a final concentration

of 1–5 mg/ml.

Note: Do not use any buffers.

Preparation of

working solution

Dilute the stock solution with methanol to a final con-

centration of 1 ?g/ml. The working solution is stable at

2–8°C, for about 6 months.

2.2Staining of monolayer cultures

Procedure Please find the protocol for the staining of monolayer

cultures in the following table.

Step Action

1Allow cultures to reach 50–70% confluence.

Note: Allowing cultures to reach confluence will impair subsequent

visualization of mycoplasmas. Cultures may be grown on coverslips in

petri dishes.

2Pour off the medium from the cells.

3Wash once with DAPI-methanol (working solution, 1 ?g/ml).

4Cover the cells with DAPI-methanol and incubate for 15 min at 37°C.

5Pour off the staining solution.

6Wash once with methanol.

7Place the inverted coverslip on a microscope slide, using glycerol or

PBS as mounting medium, avoid water.

Examine under a fluorescence microscope with 340/380 nm excita-

tion filter and LP 430 nm barrier filter (e.g. Leitz filter combination: BP

340–380, RKB 400, LP 430; Zeiss filter combination: BP 365/11, FT

395, LP 397 or BP 340–380, RKP 400, LP 430).

A total of 500 × (40 × 12.5) magnification is generally sufficient in

detecting brightly fluorescent mycoplasmas.

But best results are obtained using a 100 × oil immersion objective.

0107.102491260016

Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany

Contact and Support

To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at:

https://www.wendangku.net/doc/8410858468.html,/support

To call, write, fax, or email us, visit the Roche Applied Science home page, https://www.wendangku.net/doc/8410858468.html,, and select your home country. Country- specific contact information will be displayed. Use the Product Search func-tion to find Pack Inserts and Material Safety Data Sheets.

2.3Staining of suspension cultures

Procedure

Please find the protocol for the staining of suspension cultures in the following table.2.4Permanent preparations

Procedure

Please refer to the following table.3.Analysis

General

An uncontaminated cell culture shows only nuclear flu-orescence against a dark cytoplasmatic background. Mitochondrial DNA does bind the fluorochrome, but at levels imperceptible by routine fluorescent microscopy. Mycoplasmas, however, which have approximately 10 times the DNA content of mitochondria, are readily detected as bright foci against the dark background. They give pin points over the cytoplasm and sometimes in intercellular spaces (s. Fig.1). Not all of the cells will necessarily be infected, so most of the preparation should be carefully scanned before declaring the cul-ture uncontaminated.

To overcome problems associated with the analysis of many different cells, to detect low-level contaminations in resistant cell lines and to screen potentially infected sera it is recommended to use an indicator cell such as 3T6 mouse embryo fibroblasts, Vero monkey cells or Mv1Lu mink lung cells (10). Specimens to be analyzed are inoculated into the indicator cell culture and, after an appropriate incubation period, the indicator cell line is analyzed for the presence of mycoplasmas.

Figure 1

Fibroblast cell line L-929 after DAPI staining of DNA.a: cell culture contaminated with mycoplasmas;

b: complete absence of mycoplasmas after a 3 cycle treatment with BM-Cyclin*

(by courtesy of by Dr. J. Schmidt, Munich-Neuherberg).

Step Action 1?Spin the cells down.

?Pour off the supernatant.2Wash once in DAPI-methanol.

3Suspended the cells in DAPI-methanol (working solution, 1 ?g/ml) and incubate for 15 min at 37°C.

4Spin the cells down.5Remove the staining solution.6Add PBS just to suspend the cells.

7

Place one drop on a microscope slide, cover with a coverslip and examine under a fluorescence microscope.

Step Action 1Stain as described in 2.3.2Pour off the staining solution.

3Wash once with methanol.4Air dry.

5

Embed the preparation with a suitable anti-fad-ing mounting medium [e.g. glycerol/PBS (10:1) containing 2-7 mM 4-phenylenediamine, pH 8.5–9.0 (9)].

a

b

4. References

1Barile, M. F., Hopps, H. E. & Grabowski, M. W. (1978) In: Myco-plasma Infection of Cell Cultures (McGarrity, G. J., Murphy, D. G. & Nichols, W. W., eds.) Plenum Press, New York,

2McGarrity, G. J. (1982) In: Advances in Cell Culture, Vol. 2 (Mar-amorosch, K., eds.) Academic Press, New York, pp.99–131.

3McGarrity, G. J. & Kotani, H. (1985) In: The Mycoplasmas, Vol. IV (Razin, S. & Barile, F., eds.) Academic Press, New York, pp.353–390.

4McGarrity, G. J., Vanaman, V. & Sarama, J. (1984) In Vitro 20, 1–18.

5Rotten, S. & Barile, M.F. (1993) TIBTECH 11, 143–151.

6Russel, W. C., Newman, C. & Williamson, D. H. (1975) Nature 253, 461–462.

7Chen, T. R. (1977) Exp. Cell. Res . 104, 255–262.

8Hessling, J. J., Miller, S. E. & Levy, N. L. (1980) J. Immunol. Meth-ods 38, 315–324.

9Krenik, K. D. et al. (1989) J. Immunol. Meth . 117, 91–97.10Darai, G. et al. (1983) In Vitro 19, 7–15.

5.Changes to previous Version:

Minor editorial changes

6. Related products

*available from Roche Applied Science

Product

Pack Size Cat. No.BM-Cyclin

Antibiotic combination for the elimination of mycoplasma from cell cultures

37.5 mg

(for 2 × 2.5 l culture

medium)

10 799 050 001

Mycoplasma Detection Kit 1 kit (25 tests)11 296 744 001Mycoplasma PCR ELISA

1 kit (96 reactions)

11 663 925 001

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