baseclickfor High Throughput Screening, sensitive ClickTech EdU Cell Proliferation Kit 488 Sensitive, For 4 x 96 well plate assays. User guide

Version 4.0

User Manual

ClickTech Sensitive EdU Cell Proliferation Kit

for High-throughput Screening

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

2

Ordering information

(for detailed kit content see Table 2)

EdU DetectPro HTS Kits ready for 2 x 96 well plate assays:

Product number

EdU

Used fluorescent dye

BCK-EdUPro488HTS2

2 mL

Eterneon² GREEN Azide

(Enhancer system – incl. FITC alternative)

BCK-EdUPro555HTS2

2 mL

Eterneon² YELLOW Azide

(Enhancer system – incl. Cy3 alternative)

EdU DetectPro HTS Kits ready for 4 x 96 well plate assays:

Product number

EdU

Used fluorescent dye

BCK-EdUPro488HTS4

2 x 2 mL

Eterneon² GREEN Azide

(Enhancer system – incl. FITC alternative)

BCK-EdUPro555HTS4

2 x 2 mL

Eterneon² YELLOW Azide

(Enhancer system – incl. Cy3 alternative)

For References, FAQs and ordering please see online or contact us:

online: www.baseclick.eu

orders: [e-mail]

support: [e-mail]

phone: [phone]

fax: [phone]

ClickTech Sensitive EdU Cell Proliferation
Kit for High-throughput Screening

3

ClickTech Sensitive EdU HTS Kit

Introduction and product description:

The detection of cell proliferation is of utmost importance for assessing cell health, determining
genotoxicity or evaluating anticancer drugs. This is normally performed by adding nucleoside analogues
like [3H]thymidine or 5-bromo-2’-deoxyuridine (BrdU) to cells during replication, and their incorporation
into DNA is detected or visualized by autoradiography or with an anti-BrdU-antibody respectively. Both
methods exhibit several limitations. Working with [3H]thymidine is troublesome because of its
radioactivity. Autoradiography is slow and thus not suitable for rapid high-throughput studies. The major
disadvantage of BrdU staining is that the double-stranded DNA blocks the access of the anti-BrdU antibody
to BrdU units. Therefore, samples have to be subjected to harsh denaturing conditions resulting in
degradation of the structure of the specimen.

For research use only.

Information in this document is subject to change without notice. baseclick GmbH assumes no
responsibility for any errors that may appear in this document.

baseclick GmbH disclaims all warranties with respect to this document, expressed or implied, including but
not limited to those of merchantability or fitness for a particular purpose. In no event shall baseclick GmbH
be liable, whether in contract, tort, warranty, or under any statute or on any other basis for special,
incidental, indirect, punitive, multiple or consequential damages in connection with or arising from this
document, including but not limited to the use thereof.

Please read the material safety data sheets (MSDS) provided for each product/component.

Literature Citation: When describing a procedure for publication using this product, please refer to it as
baseclick ClickTech Sensitive EdU HTS Kit.

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

4

How the enhanced Sensitive EdU cell proliferation assay works

The baseclick Sensitive EdU overcome these limitations, providing a superior alternative to BrdU and
[3H]thymidine assays for measuring cell proliferation.

Just as in the traditional EdU proliferation kits from baseclick, also here EdU (5-ethynyl-2’-deoxyuridine) (a
nucleoside analog to thymidine) is incorporated into DNA during active DNA synthesis. In contrast to BrdU
assays, the Sensitive EdU are not antibody based and therefore do not require DNA denaturation for
detection of the incorporated nucleoside. Instead, the Sensitive EdU utilize click chemistry for detection
in a variety of dye fluorescent readouts. Furthermore, the streamlined detection protocol reduces both
the total number of steps and significantly decreases the total amount of time.

The simple click chemistry detection procedure is complete within 30 minutes and is compatible with
multiplexing for content and context-rich results.

Figure 1: Comparison of the fluorescence intensities between the standard EdU Kit and the Sensitive EdU Kit.

ClickTech Sensitive EdU Cell Proliferation
Kit for High-throughput Screening

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The baseclick Sensitive EdU HTS Kit can be used with antibodies against surface and intracellular markers.
To ensure the compatibility of your reagent or antibody, please refer to Table 1.

Table 1: EdU detection dye compatibility

Fluorescent molecule

Compatibility*

Organic dyes such as Fluorescein and Alexa
dyes

Compatible

PerCP, Allophycocyanin (APC) and APC-based
tandems

Compatible

R-phycoerythrin (R-PE) and R-PE based
tandems

Use R-PE and R-PE based tandems after the EdU
detection reaction

Quantum Dots

Use Quantum Dots after the EdU detection reaction

Fluorescent proteins (e.g. GFP)

Use anti-GFP antibodies** before the EdU detection
reaction or use organic dye-based reagents for
protein expression detection

* Compatibility indicates which involved components are unstable in the presence of copper catalyst for
the EdU detection reaction (either the fluorescent dye itself or the detection method).

** The resulting fluorescence intensity depends strongly on the antibody manufacturer and target.
Internal tests have shown a generally good fluorescence amount for rabbit and chicken anti-GFP and a
very low fluorescence amount for mouse monoclonal antibodies. This can be understood as a general
guideline but results may still greatly vary depending on the individual chosen antibody.

Cautions:

The rinse buffer (Component R): contains hazardous components. Use with appropriate precautions. Keep
away from acids to avoid dangerous gases.

Handle reagents containing the rinse buffer using equipment and practices appropriate for
the hazards posed by such materials. Use gloves. Dispose of the reagents in compliance with all related
local arrangements.

This solution is stored at RT and will crystallize at lower temperatures. If crystallized, the solution has to
be brought to RT, mixed thoroughly and can then, once homogenously dissolved, be used without further
considerations. The activity of this compound is not affected hereby.

MSDS: the appropriate MSDS can be downloaded from our website www.baseclick.eu.

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

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1. Materials provided with the Kit and storage conditions

Table 2: Contents of the kit and storage conditions

Color code

Amount for 2

assays/well

plates

Amount for 4

assays/well

plates

Component

Component

long term

storage

Kit short

term

storage*

Component E

yellow

2 mL

2 x 2 mL

5-Ethynyl-deoxyuridine
(5-EdU)

– 20 °C

2 - 8 °C

Dark

Do not

freeze

Dry

Component D

red

1 x 60 µL

2 x 60 µL

• Eterneon

2

GREEN

Azide (BCK-EdUPro­HTS488)

• Eterneon

2

YELLOW

Azide (BCK-EdUPro­HTS555)

– 20 °C

dark

Component

RP

40 mL

40 mL

Reaction Buffer

2 - 8 °C

Component C

green

1 mL

2 mL

Reactor System

2 - 8 °C

Component B

200 mg

2 x 200 mg

Buffer additive

2 - 8 °C/

– 20 °C**

Component R

6 mL

2 x 6 mL

Rinse buffer (10x)

RT

* This kit is stable up to 1 year after receipt, when stored as directed.

** When dissolved the component has to be kept at – 20 °C for long-term storage. Prepare aliquots to
avoid too many freeze and thaw cycles; if the solution starts to develop a brown colour, it has degraded
and should be discarded.

2. Required Material and Equipment not included in this kit

• Adherent cells

• Reaction tubes (size depends on the volume of reaction cocktail needed)

• Buffered saline solution, such as PBS, D-PBS or TBS

• Fixative solution (4% Paraformaldehyde in PBS)

• Permeabilization solution optimised for your cell line (for example, 0.5% Triton® X-100 in PBS, or

a 0.5% saponin-based solution)

• Appropriate cell culture medium

• 1% BSA (bovine serum albumin) in PBS, pH 7.1 - 7.4

• Deionized water or 18 MΩ purified water

ClickTech Sensitive EdU Cell Proliferation
Kit for High-throughput Screening

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3. Workflow

The following protocol was developed using a final EdU concentration of 10 µM and can be adapted for
any cell type. There are many factors, which can influence the labeling such as the growth medium, the
density and the type of cells. To determine the optimal concentration for your experiment, a range of EdU
concentrations should be tested for your cell type and experimental conditions.

Principally, a similar concentration to BrdU can be used for EdU as a starting point. Heparin can be used as
anticoagulant for collection, if a whole blood sample is used.

Workflow scheme for the Sensitive EdU HTS Assay

Incubate sample with EdU

↓

Harvest cells

↓

Optional: Treat cells with antibodies to cell surface antigens

↓

Fix and permeabilize cells*

↓

Optional: Treat cells with antibodies to intracellular antigens

↓

Detect / Label EdU

↓

Wash cells well with rinse buffer

↓

Optional: Treat cells with cell cycle stain

↓

Imaging and Analyse Cells

* At this point the sample can be stored safely

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

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4. Preparation of the stock solutions

4.1. Allow all vials to warm to room temperature before opening.

4.2. For the preparation of a 20 µM stock solution of EdU (2x EdU), add the appropriate amount of

aqueous solution (1x PBS) to EdU (component E) according to Table 3 and mix until the compound
is completely dissolved. After use, store any remaining solution at – 20 °C. When stored as
directed, this stock solution is stable for up to one year.

Table 3: Amounts of aqueous solution needed to dissolve EdU to a final concentration of 20 µM

EdU DetectPro HTS Kit

20X EdU solution

Dilution volume of 1x PBS

2 x 96 well plates

2 mL

18 mL

4 x 96 well plates

4 mL

36 mL

4.3. For the preparation of a stock solution of the buffer additive, add the appropriate amount of

deionized water (see Table 4) to the component B and mix until the compound is dissolved
completely. After use, store any remaining solution at – 20 °C. When stored as directed, this stock
solution is stable for up to 6 months. We recommend preparing aliquots to avoid repeated freeze
and thaw cycles!

Table 4: Amounts of aqueous solution needed to dissolve the buffer additive to the final work solution

EdU DetectPro HTS Kit

Buffer additive (solid)

Dilution volume of deionized water

2 x 96 well plates

200 mg

2.5 mL

4 x 96 well plates

400 mg

5.0 mL

ClickTech Sensitive EdU Cell Proliferation
Kit for High-throughput Screening

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5. Labeling of cells with EdU

5.1. Suspend the cells in an appropriate tissue culture medium to obtain optimal cell growth

conditions. Please note that the growth of the cells during incubation decelerates, if the
temperature changes or the cells are washed prior to incubation with EdU.

5.2. For the desired final concentration, add the appropriate amount of EdU to the culture medium

and mix well. We recommend using a concentration of 10 µM for 1 - 4 hours as a starting point.
Use higher EdU concentrations for a shorter incubation time. A longer incubation time requires
lower EdU concentrations.

5.3. The incubation of the cells with EdU should be performed under the optimal conditions for your

cell type, the number of cells plated and for the desired length of time. Various DNA synthesis
and proliferation parameters can be evaluated by altering the EdU incubation time or by
subjecting the cells to pulse labeling with EdU. Effective time intervals for pulse labeling and the
length of each pulse depend on the cell growth rate and the number of cells used.

5.4. If performing antibody surface labeling, proceed immediately to step 6, otherwise continue to

step 7.

6. Staining cell-surface antigens with antibodies (optional)

6.1. Wash cells in each well with 100 µL of 1% BSA in PBS.

6.2. Remove the wash solution and add again 100 µL of 1% BSA in PBS to the cells.

6.3. Add surface antibodies and mix well but gently.

Note: PE, PE-tandem or Quantum Dot antibody conjugates should not be used before performing
the click reaction (step 8).

6.4. Incubate the cells for the recommended length of time and temperature. Protect from light!

6.5. Proceed to step 7.

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

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7. Cell fixation and permeabilization

This protocol was developed with a fixation step using 4% Paraformaldehyde in PBS, followed by
permeabilization step. A saponin-based permeabilization solution can be used with cell samples containing
red blood cells or whole blood as well as with cell probes containing different cell types. The morphological
light scatter characteristics of leukocytes are maintained by a saponin-based solution while red blood cells
are lysed.

7.1. Remove the incubation media and wash the cells, each well with 100 µL of 1% BSA in PBS.

Afterwards remove the wash solution.

7.2. Add 100 µL of the fixative solution to the cells in each well. Incubate for 15 minutes at room

temperature. Protect from light.

7.3. Remove the fixation solution and wash the cells in each well twice with 200 µL of 1% BSA in PBS.

If red blood cells or haemoglobin are present in the sample, repeat the washing step. Remove all
residual blood cell debris and haemoglobin before proceeding.

NOTE: At this point of the procedure, the probes can be stored safely.

7.4. Remove the wash solution and add to each well 100 µL of permeabilization solution. Mix well

but gently, incubate for 20 minutes at room temperature and proceed to step 8. for the click
reaction.

8. EdU detection

8.1. Prepare the click assay cocktail in the same order as described in Table 5. If the ingredients are

not added in the order listed, the reaction will not proceed optimally or might even fail.

Important: Once the assay cocktail is prepared, use it immediately, at least within the next
15 minutes!

Table 5: Click assay cocktails

Material

Component code

Number of 96 well plates

1 2 4

Reaction buffer

RP

9.447 mL

18.89 mL

37.78 mL

Reactor system

C - green

440 µL

880 µL

1760 µL

Dye Azide

D - red

23 µL

46 µL

92 µL

Buffer additive

(prepared in 4.3)

B

1.1 mL

2.2 mL

4.4 mL

Total Volume

-

11.01 mL

22.02 mL

44.04 mL

ClickTech Sensitive EdU Cell Proliferation
Kit for High-throughput Screening

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8.2. Remove permeabilization solution from step 7.4 and add 100 µL of the click assay cocktail to each

well and mix well but gently to distribute the assay solution evenly.

8.3. Incubate the click assay mixture for 30 minutes at room temperature. Protect from light!

8.4. From the 10x rinse solution prepare a 1x rinse solution by applying following table (Table 6). Add

the appropriate amount of PBS (1x) (see Table 6) to the component R and mix well (To prevent
crystallization, keep component R at room temperature at all times. If component R has

crystalized, please warm up to dissolve again. Please see also “cautions”). This additional wash

step with this special rinse buffer reduces unspecific, cell number dependent background signal.
After use, store any remaining solution at RT. When stored as directed, this stock solution is stable
for up to 6 months.

Table 6: Amounts of aqueous solution needed to dissolve the rinse buffer to the final work solution

Sensitive EdU HTS Kit

Volume of 10x rinse buffer

Dilution volume of 1x PBS

2 x 96 well plates

5.8 mL

52.2 mL

4 x 96 well plates

11.5 mL

103.5 mL

Remove Click assay cocktail and wash the cells in each well twice with 150 µL with the 1x rinse
solution prepared above.

8.5. Remove rinse solution. 100 µL of 1% BSA in PBS is then given to the cells in each well.

8.6. If performing antibody surface or intracellular labeling, proceed immediately to step 9, otherwise

continue to step 10.

ClickTech Sensitive EdU Cell Proliferation

Kit for High-throughput Screening

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9. Staining intracellular or surface antigens (optional)

9.1. Add antibodies against intracellular antigens or against surface antigens that use RPE, PR-tandem

or Quantum Dot antibody conjugates. Mix well.

9.2. Incubate the cells for the time and temperature required for antibody staining. Protect from light.

9.3. Wash each well twice with 100 µL permeabilization solution. Remove the solution. Add again

100 µL of 1% BSA in PBS to the cells.

9.4. Proceed with step 10 for analysing the cells.

10. Imaging and analysis

10.1. Close the 96 well plate by using a sealing film, if desired.

10.2. Fluorescence is quantified by scanning the plate using an automated imaging platform equipped

with filters appropriate for the dye used. Images of each well can be taken by microscopy.
The excitation and emission maxima of the available dyes are listed in Table 7.

Table 7: Emission and excitation maxima of the available dyes.

Product number

Dye

Excitation

(nm)

Emission

(nm)

Filter

BCK-EdUPro488HTS2

-----------------------------­BCK-EdUPro488HTS4

Eterneon2 GREEN Azide

(Enhancer system – incl. FITC alternative)

496

516

green

BCK-EdUPro555HTS2

-----------------------------­BCK-EdUPro555HTS4

Eterneon2 YELLOW Azide

(Enhancer system – incl. Cy3 Azide

alternative)

546

579

yellow