Lonza_ManualsProductInstructions_MycoAlert_Mycoplasma_Detection

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Document # INST-LT07-118 04/11

MycoAlert™ mycoplasma detection kit

Mycoplasma detection assay - Instructions for use

Safety

THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not

approved for human or veterinary use, for application to humans

or animals, or for use in clinical or in vitro procedures.

LT07-218

MycoAlert™ reagent

(lyophilized)

MycoAlert™ assay buffer

MycoAlert™ substrate

(lyophilized)

25 tests

5 x 600 µl (LT27-217)

1 x 10 ml (LT27-218)

5 x 600 µl (LT27-221)

1. MycoAlert™ assay procedure outline

(For detailed assay protocol see section 9)

Spin 2 ml cell sample at 200 x g for 5 min.

Remove 100 µl of cleared supernatant to fresh

tube/well

Reconstitute MycoAlert™ reagent and

MycoAlert™ substrate in assay buffer

Equilibration time 15 minutes.

Add 100 µl MycoAlert™ reagent to sample.

Wait for 5 minutes.

Measure luminescence.

(Reading A)

Add 100 µl MycoAlert™ substrate to sample.

Wait for 10 minutes.

Measure luminescence.

(Reading B)

2. Kit contents & ordering information

LT07-418

MycoAlert™ reagent

(lyophilized)

MycoAlert™ assay buffer

MycoAlert™ substrate

(lyophilized)

50 tests

2 x 2.5 ml (LT27-237)

1 x 10 ml (LT27-218)

2 x 2.5 ml (LT27-238)

LT07-318

MycoAlert™ reagent

(lyophilized)

MycoAlert™ assay buffer

MycoAlert™ substrate

(lyophilized)

100 tests

1 x 10 ml (LT27-216)

1 x 20 ml (LT27-220)

1 x 10 ml (LT27-224)

Part codes in parenthesis cannot be ordered as separate item.

Related products:

MycoAlert™ assay control set

LT07-518 10 Tests

3. Storage conditions

Kit components

Store at 2˚C-8˚C. Do not freeze.

See kit label for expiry date of the whole kit. See

bottle labels for expiry dates of individual

components.

Reconstituted

Only keep reconstituted components at room

reagent and/or

temperature* for the course of the experiment.

substrate

Unused components can be aliquoted and stored at

-20°C up to six months. When using a frost-free

freezer, be aware that temperature does not keep

-20°C constantly. For improved stability we would

recommend storage at -80°C.

Once thawed, reagent and/or substrate must not be

refrozen and should be allowed to reach room

temperature before use without the aid of artificial

heat.

* If room temperature is > 30°C it might be advisable to use a water bath

set to 20°C.

LT07-118

MycoAlert™ reagent

(lyophilized)

MycoAlert™ assay buffer

MycoAlert™ substrate

(lyophilized)

10 tests

2 x 600 µl (LT27-217)

1 x 10 ml (LT27-218)

2 x 600 µl (LT27-221)

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Page 1 of 7

This increase in ATP can be detected using the

following bioluminescent reaction:

4. Intended use

Mycoplasma are the smallest and simplest

prokaryotes. Mycoplasma depend on their hosts for

many nutrients due to their limited biosynthetic

capabilities. They have long been recognized as

common contaminants of cells in continuous culture

but their presence may go undetected for months.

As the mycoplasma competes with the cells for the

nutrients in culture media, one of the first signs is a

reduction in the rate of cell proliferation and slight

changes in cellular responses including gene

expression.

Luciferase

ATP+ Luciferin + O

2

Oxyluciferin + AMP + PP

i

+ CO

2

+ LIGHT

Mg

2+

The emitted light intensity is linearly related to the

ATP concentration and is measured using a

luminometer. The assay is conducted at room

temperature (18°C-22°C), the optimal temperature

for luciferase activity.

1000.0

M. orale

M. hyorhinis

A. laidlawii

100.0

Mycoplasma detection in cell cultures has until now

been a long, drawn out process with difficult-to-

interpret results. The MycoAlert™ kit is intended for

the quick and convenient detection of viable

mycoplasma in cell cultures. The speed and

convenience of the MycoAlert™ kit allows for the

routine testing of cells in culture and commonly used

constituents of complete media.

To allow for the early detection of mycoplasma

contamination Lonza recommends testing at every

cell passage. Frequent testing such as this will

indicate when a cell line becomes infected allowing

prompt remedial action to be taken. The MycoAlert™

assay can also be extended to incoming cell lines

and the commonly used constituents of complete

media.

R

a

t

i

o

10.0

1.0

3206401280

CFU per ml

0.1

Figure 1: The graph shows a dilution series of M.

hyorhinis, M. orale and A. laidlawii demonstrating the

sensitivity of the MycoAlert™ assay.

MycoAlert - K562 Cell Culture

5. Assay principle

The MycoAlert™ assay is a selective biochemical

test that exploits the activity of certain mycoplasmal

enzymes. The presence of these enzymes provides

a rapid screening procedure, allowing sensitive

detection of contaminating mycoplasma in a test

sample (figure 1). The viable mycoplasma are lysed

and the enzymes react with the MycoAlert™

substrate catalyzing the conversion of ADP to ATP.

By measuring the level of ATP in a sample both

before and after the addition of the MycoAlert™

substrate, a ratio can be obtained which is indicative

of the presence or absence of mycoplasma (figure

2). If these enzymes are not present, the second

reading shows no increase over the first, while

reaction of mycoplasmal enzymes with their specific

substrates in the MycoAlert™ substrate, leads to

elevated ATP levels.

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

R

L

U

10000

M. hyorhinis infected : ratio 114

Negative control : ratio 0.91

1000

Reading A

100

Reading B

10

1

02468

Time (minutes)

10121416

Figure 2: The kinetics of ATP generation in the presence

of nis contamination.

Page 2 of 7

Beta counters: Mode - out of coincidence or

luminescence; read time 1 second (integrated).

6. Component reconstitution and storage

NOTE: Please read this section carefully to ensure optimal

performance for your assay. Ensure that you follow the

correct component reconstitution applicable to the kit

size you have received (see table below).

The MycoAlert™ reagent and substrate are supplied

as lyophilized pellets. These are reconstituted in the

supplied MycoAlert™ assay buffer to produce the

working solutions for use in the assay.

1. For reconstitution add MycoAlert™ buffer to the

MycoAlert™ reagent and substrate, according to

the table below (volumes depend on kit size).

MycoAlert™

assay buffer added

LT07-118 (10 test kit)

LT07-218 (25 test kit)

LT07-418 (50 test kit)

LT07-318 (100 test kit)

b. Additional equipment and consumables

- 10 ml sterile pipettes

- Luminometer cuvettes or white walled

microplates (ideally with an opaque bottom)

- Micropipettes: 50-200 µl; 200-1000 µl

- Bench centrifuge

8. Assay samples & controls

Following sample types are suitable for use with

MycoAlert™ assay. Cell supernatant must be spun

at 1500 rpm (200 x g) for 5 minutes to remove any

remaining cells. Cells present in the sample will

increase the background, resulting in the loss of

sensitivity and resolution. In essence it reduces the

chance of seeing a low-lying infection.

a. Fresh supernatant (optimal sample)

1. Cell supernatant during passage of

suspension cell culture.

2. Supernatant from adherent cells prior to

trypsinisation.

3. Cell supernatant from cells brought “up from

frozen” – cells out of liquid nitrogen with

uptake into media. Leave minimum of 1-2hr

before testing under normal culture

conditions.

NOTE: Cells diluted into fresh media after passaging or

trypsinization result in a much lower signal - leave

minimum 24hr under normal culture conditions before

testing.

to

MycoAlert™

reagent

600 µl

2.5 ml

10 ml

to

MycoAlert™

substrate

600 µl

2.5 ml

10 ml

2. Replace screw cap and mix gently.

3. Allow equilibration to room temperature for at

least 15 min.

4. Only keep reconstituted components at room

temperature for the course of the experiment.

Please refer to section 3 for longer term storage.

7. Additional equipment

a. Instrumentation

The MycoAlert™ kit requires the use of a

luminometer. The assay has been designed for use

with cuvette/tube and/or plate luminometers. Lonza

provides a list of luminometers which have been

tested for compatibility with the MycoAlert™ assay

(/luminometers). If your

luminometer is not on that list we highly recommend

assessing its sensitivity using our MycoAlert™ assay

control set (LT07-518).

The parameters of the luminometer should be

assessed and the conditions below used to produce

the correct programming of the machine.

Cuvette/tube luminometers: Read time 1 second

(integrated).

Plate luminometers: Read time 1 second

(integrated). When using a microplate reader all

reagents should be added manually.

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Supernatant can be stored at room temperature or

4°C for testing the same day.

b. Refrigerated supernatant

Supernatants might be stored at 4°C for ≤ 5 days.

Bring to room temperature without the aid of artificial

heat before testing. Do not freeze supernatant.

c. Other samples suitable for MycoAlert™:

•

Cell free media

•

FCS

•

BPE

•

Other tissue culture reagents

d. Positive control

A MycoAlert™ positive control is available as

separate item (LT07-518). This control does not

contain mycoplasma, i.e. it is not a source of

Page 3 of 7

mycoplasma. We recommend to include a positive

control sample in every experiment.

e. Negative control

Use 100 µl of MycoAlert™ buffer or HPLC grade

water as a negative control. We recommend to

include a negative control sample in every

experiment.

However, the test has been designed to give ratios

of less than 0.9 with uninfected cultures. Cells which

are infected with mycoplasma will routinely produce

ratios greater than 1.

Table 1. Interpretation of MycoAlert™ assay results

illustrating examples of healthy and infected cell lines.

Cell Line

Infected cells

K562

A549

U937

HepG2

Healthy cells

HL60

COS-7

MycoAlert™

ratio

123.26

4.10

8.26

1.17*

0.72

0.46

Conclusions

Positive

Borderline,quarantine

and retest in 24 hours

Negative

9. Assay protocol

NOTE: To ensure that the optimal performance of the

assay is achieved for your experiment please

make certain that you have carefully read the

component reconstitution and storage procedure.

1. Bring all reagents up to room temperature

before use.

2. Reconstitute the MycoAlert™ reagent and

MycoAlert™ substrate in MycoAlert™ assay

buffer. Leave for 15 minutes at room

temperature to ensure complete rehydration.

3. Transfer 2 ml of cell culture or culture

supernatant into a centrifuge tube and pellet any

cells at 1500 rpm (200 x g) for 5 minutes.

4. Transfer 100 µl of cleared supernatant into a

luminometer cuvette/tube or well.

5. Program the luminometer to take a 1 second

integrated reading (this is usually the default

setting on most cuvette luminometers).

6. Add 100 µl of MycoAlert™ reagent to each

sample and wait 5 minutes.

7. Place cuvette or plate in luminometer and initiate

the program (Reading A).

8. Add 100 µl of MycoAlert™ substrate to each

sample and wait 10 minutes.

9. Place cuvette or plate in luminometer and initiate

the program (Reading B).

10. Calculate ratio = Reading B/Reading A.

* see Troubleshooting section 11

11. Troubleshooting

High background levels?

Take great care when handling any of the reagents.

Skin has high levels of ATP on its surface that can

contaminate the reagents leading to falsely high

readings. Wear latex gloves or equivalent.

Ensuring optimal performance

The optimal working temperature for all reagents is

22°C. If reagents have been refrigerated always

allow time for them to reach room temperature

before use.

Pipettes

As with all assays involving manual pipetting in order

to gain maximal accuracy and to reduce variability

pipettes should be calibrated regularly.

Borderline ratios around 1

The sensitivity of the assay does allow for detection

of covert contamination, and if the ratio is marginally

above 1 (for example up to 1.2), it is recommended

that the sample be retested. Any cultures maintained

in quarantine can be tested after a further 24-48

hours in culture to see if the ratios have increased.

A ratio of less than 1 is produced by the ongoing

consumption of ATP over the time course of the

assay. Consistent ratios of around 1 demonstrate

that this consumption and subsequent drop in RLUs

is not being seen and indicates an instrument

sensitivity issue.

10. Interpretation of results

The ratio of Reading B to Reading A is used to

determine whether a cell culture is contaminated by

mycoplasma.

Ratio

< 0.9

0.9 - 1.2

> 1.2

Interpretation

Negative for mycoplasma

Borderline: quarantine cells & retest

in 24 h

Mycoplasma contamination

The interpretation of the different ratios obtained,

within each experimental situation, may vary

according to the cell types and conditions used.

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Page 4 of 7

To try to overcome this, increase the integration

time from 1 second up to a max of 10 seconds;

check to make sure that filters (not even plain

glass) are not present between the sample and

detector, and ensure the instrument is in

luminescence or “out of coincidence” mode.

Negative RLUs or ratios

If automatic background subtraction is enabled on

the instrument it will cause negative RLUs for the B

reading and consequently negative ratios. This

option MUST be disabled for the instrument to work

correctly with the MycoAlert™ assay.

If technical support is required please contact Lonza

Scientific Support teams.

Mycoplasma equirhinis

Mycoplasma faucium

Mycoplasma fermentans

Mycoplasma gallinaceum

Mycoplasma gallisepticum

Mycoplasma genitalium

Mycoplasma hominis

Equine

Human

Positive

Mammalian/Avian Positive

Avian

Human

Positive

Mycoplasma hyopneumoniae Human

Mycoplasma hyorhinis

Mycoplasma hyosynoviae

Mycoplasma iguanae

Mycoplasma lipophilum

Mycoplasma muris

Mycoplasma neurolyticum

Mycoplasma opalescens

Mycoplasma orale

Mycoplasma pirum

Mycoplasma pneumoniae

Mycoplasma primatum

Mycoplasma pulmonis

Mycoplasma salivarium

Porcine

Iguana

Human

Murine

Canine

Human

Mammalian

Human

Rat

Human

12. MycoAlert™ tested species

The MycoAlert™ mycoplasma detection kit is a

generic biochemical test for mycoplasma and other

mollicutes (e.g. acholeplasmas, mesoplasmas,

spiroplasmas) and will detect mollicutes of

mammalian, avian, insect and plant origin.

The following 44 mollicute species were tested using

the MycoAlert™ assay. Species were obtained from

the National Collection of Type Cultures UK. The six

most common species in cell culture are in bold.

Species

Acholeplasma laidlawii

Acholeplasma modicum

Acholeplasma morum

Origin/Source Result

Mycoplasma spermatophilum Human

Mycoplasma synoviae

Spiroplasma citri

Avian

Plant/Insect

13. Preventing mycoplasma contamination

Mammalian/Avian Positive

Bovine

Mammalian

Positive

Mesoplasma entomophilum Insect

Mesoplasma florum

Mycoplasma agussizii

Mycoplasma alkalescens

Mycoplasma alligatoris

Mycoplasma arginini

Mycoplasma arthriditis

Mycoplasma bovirhinis

Mycoplasma bovis

Mycoplasma bovoculi

Mycoplasma buccale

Mycoplasma californicum

Mycoplasma canadense

Mycoplasma cloacale

Mycoplasma conjunctivae

Mycoplasma crocodyli

Plant/insect

Tortoise

Bovine

Alligator

Bovine/Porcine

Human

Bovine

Human

Bovine

Avian

Ovine & Caprine

Crocodile

Mycoplasmas, the smallest and simplest form of

bacteria, are common contaminants of cells grown in

culture. Studies indicate that between 15% - 35% of

all continuous culture cells are contaminated with

mycoplasma (Rottem and Barile, 2003). Infections

can seriously impact the reliability, reproducibility

and consistency of results obtained with these

cultures, and can be easily spread within the culture

environment. To that end we recommend aseptic

techniques to prevent mycoplasma contamination

and cross contamination with other cell lines.

•

Wear appropriate personal protective

equipment (sterile gloves, lab coat, safety

glasses).

•

Perform all tissue culture work in a biosafety

cabinet at appropriate containment level.

•

Sanitize the biosafety cabinet with 70%

ethanol before commencing work.

•

Wash gloved hands with 70% ethanol and

allow to air dry for 30 seconds.

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Page 5 of 7

o

If gloves are contaminated by

touching anything outside the

cabinet, re-sanitize as above.

o

Discard gloves after handling

contaminated cultures and at the

end of all culture procedures.

Use 70% ethanol to disinfect exterior

surfaces of all materials and equipment

required for experiment before placing in to

the biosafety cabinet.

Ensure air flow in the biosafety cabinet

circulates properly.

o

Direct verbal communication away

from the cabinet.

o

Minimize rapid movement within and

immediately outside the cabinet.

Use sterile flasks, plates, bottles and dishes

for all cell cultures and media.

Dedicate separate media for each cell line.

Minimize exposure of sterile media, cell

cultures, and equipment to the environment.

o

Uncover sterile culture vessels

immediately before use; re-cover as

soon as work is finished.

o

Keep sterile lab equipment

(pipettes, reservoirs, plates, etc)

wrapped until ready to use.

o

Return cultures to incubator as soon

as work is complete.

Avoid splashes, spills, and aerosols.

Do not transfer liquid by pouring; use a new,

sterile pipette for each transfer to or from a

different bottle.

Cleanup after tissue culture work is

complete.

o

Disinfect all equipment and material

with 70% ethanol before removing

from cabinet.

o

Disinfect work surfaces inside of

biosafety cabinet with 70% ethanol.

Use the MycoAlert™ mycoplasma

detection kit to routinely screen cell

cultures.

Miller,J., Kassem,S., Pepper,S.D., Hey,Y., Ward, T. H., and

Margison,G.P. (2003) Mycoplasma infection significantly alters

microarray gene expression profiles. Biotechniques, 35(4): 812-

814.

Razin,S., Yogeu,D, and Naot,Y: (Dec 1998) Moleculer Biology

and Pathogenicity of Mycoplasmas. Microbiol and Molecular

Biology Reviews, 1094-1156.

Rowe,J.A., Scragg,I., Kwiatkiwski,D., Ferguson,D., Carucci,D.,

and Newbold,C. (1998) Implications of mycoplasma

contamination in Plasmodium falciparum cultures and methods for

its detection and eradication. Molecular and Biochemical

Parasitology, 92: 177-180.

Rottem, S. and Barile, M.F. (1993). Beware of Mycoplasma.

Trends in Biotechnology, 11(4): 143-151.

•

MycoAlert™ citations:

Jian-Zhong Qin, Lawrence Stennett, Patricia Bacon, Barbara

Bodner, Mary J.C. Hendrix, Richard E.B. Seftor, Elisabeth A.

Seftor, Naira V. Margaryan, Pamela M. Pollock, Amy Curtis,

Jeffrey M. Trent, Frank Bennett, Lucio Miele, and Brian J.

Nickoloff. (2004). p53-independent NOXA induction overcomes

apoptotic resistance of malignant melanomas. Molecular Cancer

Therapy, 3: 895-902.

Yong Zhou, James S. Hagood and Joanne E. Murphy-Ullrich.

(2004).

Thy-1 Expression Regulates the Ability of Rat Lung

Fibroblasts to Activate Transforming Growth Factor-ß in

Response to Fibrogenic Stimuli American Journal of Pathology,

165: 659-669.

•

Product warranty

When used according to the preceding protocol

Lonza’s MycoAlert™ assay will provide a sensitive

measure of mycoplasma infection in cell cultures. It

is intended as a presumptive screening tool, and any

positives should be re-tested by a second

confirmatory method.

Lonza warrants that this product will perform

according to established product specifications. It is

sold with the understanding that the purchaser will

determine if the product is suitable for his or her

application. Lonza shall not be liable for any

damages or injury to persons or property arising

from the purchase or use of the product.

•

Lonza strongly recommends that cell cultures with

mycoplasma contamination be discarded and fresh

stocks obtained. When that’s not possible,

MycoZap™ elimination reagent provides a reliable

method of mycoplasma elimination.

References

Mycoplasma:

Denecke,J., Becker,K., Jurgens,H., Reinhold,G., and

Wolff,J. (1999) Falsification of Tetrazolium Dye (MTT) Based

Cytotoxicity Assay Results due to Mycoplasma Contamination of

Cell Cultures. Anticancer Reseach, 19: 1245-1248.

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Page 6 of 7

THESE PRODUCTS ARE FOR RESEARCH USE ONLY.

Not approved for human or veterinary use, for application to humans or

animals, or for use in clinical or in vitro procedures.

The MycoAlert™ and kits are protected by a granted patent in Australia

[AU2004233290(B2)], Brazil [BRPI0407750(A)], Canada [CA2512636(A1)],

China [CN1756848(B)], Europe [EP1613763(A1) and EP2264181(A1)], the

UK [GB2400659B], Japan [JP2006523450], South Korea

[KR2(A)], Norway [NO20053276(A)], the US [US7585644], and

the World Intellectual Property Organization [WO2004094656(A1)].

All trademarks herein are marks of Lonza Group or its subsidiaries.

Developed by Lonza Nottingham, Ltd.

Page 7 of 7

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