baseclickfor Flow Cytometry, in vivo ClickTech EdU Cell Proliferation Kit 488 in vivo, S (50 mg additional EdU), ClickTech EdU Cell Proliferation Kit 594 in vivo, M (500 mg additional EdU), ClickTech EdU Cell Proliferation Kit 488 in vivo, S (50 mg additional EdU), ClickTech EdU Cell Proliferation Kit 594 in vivo, S (50 mg additional EdU), ClickTech EdU Cell Proliferation Kit 647 in vivo, M (500 mg additional EdU) User guide

Version 6.0

User Manual

EdU in vivo Kits

ClickTech EdU Cell Proliferation

in vivo Kits

2

Ordering information

ClickTech EdU in vivo Kits for Imaging (IM):

Product number

Basic product

Additional

EdU

Dye

BCK-EdU488IM100+IV-S

BCK-EdU488IM100

50 mg

6-FAM-Azide

BCK-EdU555IM100+IV-S

BCK-EdU555IM100

50 mg

5-TAMRA-PEG3-Azide

BCK-EdU594IM100+IV-S

BCK-EdU594IM100

50 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647IM100+IV-S

BCK-EdU647IM100

50 mg

Eterneon Red 645 Azide

BCK-EdU488IM100+IV-M

BCK-EdU488IM100

500 mg

6-FAM-Azide

BCK-EdU555IM100+IV-M

BCK-EdU555IM100

500 mg

5-TAMRA-PEG3-Azide

BCK-EdU594IM100+IV-M

BCK-EdU594IM100

500 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647IM100+IV-M

BCK-EdU647IM100

500 mg

Eterneon Red 645 Azide

BCK-EdU488IM100+IV-L

BCK-EdU488IM100

1000 mg

6-FAM-Azide

BCK-EdU555IM100+IV-L

BCK-EdU555IM100

1000 mg

5-TAMRA-PEG3-Azide

BCK-EdU594IM100+IV-L

BCK-EdU594IM100

1000 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647IM100+IV-L

BCK-EdU647IM100

1000 mg

Eterneon Red 645 Azide

ClickTech EdU in vivo Kits for Flow Cytometry (FC):

Product number

Basic product

Additional

EdU

Dye

BCK-EdU488FC50+IV-S

BCK-EdU488FC50

50 mg

6-FAM-Azide

BCK-EdU555FC50+IV-S

BCK-EdU555FC50

50 mg

5-TAMRA-PEG3-Azide

BCK-EdU594FC50+IV-S

BCK-EdU594FC50

50 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647FC50+IV-S

BCK-EdU647FC50

50 mg

Eterneon Red 645 Azide

BCK-EdU488FC50+IV-M

BCK-EdU488FC50

500 mg

6-FAM-Azide

BCK-EdU555FC50+IV-M

BCK-EdU555FC50

500 mg

5-TAMRA-PEG3-Azide

BCK-EdU594FC50+IV-M

BCK-EdU594FC50

500 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647FC50+IV-M

BCK-EdU647FC50

500 mg

Eterneon Red 645 Azide

BCK-EdU488FC50+IV-L

BCK-EdU488FC50

1000 mg

6-FAM-Azide

BCK-EdU555FC50+IV-L

BCK-EdU555FC50

1000 mg

5-TAMRA-PEG3-Azide

BCK-EdU594FC50+IV-L

BCK-EdU594FC50

1000 mg

5/6-Sulforhodamine 101-PEG3-Azide

BCK-EdU647FC50+IV-L

BCK-EdU647FC50

1000 mg

Eterneon Red 645 Azide

ClickTech EdU in vivo Kits for High-throughput Screening (HTS):

Product number

Basic product

Additional

EdU

Dye

BCK-EdU488HTS2+IV-S

BCK-EdU488HTS2

50 mg

6-FAM-Azide

BCK-EdU555HTS2+IV-S

BCK-EdU555HTS2

50 mg

5-TAMRA-PEG3-Azide

BCK-EdU488HTS2+IV-M

BCK-EdU488HTS2

500 mg

6-FAM-Azide

BCK-EdU555HTS2+IV-M

BCK-EdU555HTS2

500 mg

5-TAMRA-PEG3-Azide

BCK-EdU488HTS2+IV-L

BCK-EdU488HTS2

1000 mg

6-FAM-Azide

BCK-EdU555HTS2+IV-L

BCK-EdU555HTS2

1000 mg

5-TAMRA-PEG3-Azide

ClickTech EdU Cell Proliferation
in vivo Kits

3

Generally, one of the following standard dyes is delivered with the kits:

Dye

Absorption/emission

ε

Analogous dyes

6-FAM-Azide

(

abs

=496/em=516)

83.000 cm-1M-1

Alexa 488, DyLight488,
FluorX, ATTO488

5-TAMRA-PEG3-Azide

(

abs

=546/em=579)

91.000 cm-1M-1

Alexa 555, Cy3

5/6-Sulforhodamine 101­PEG3-Azide

(

abs

=584/em=603)

116.000 cm-1M-1

Texas Red, Alexa 594,
ATTO 594

Eterneon Red 645 Azide

(

abs

=643/em=662)

250.000 cm-1M-1

Alexa 647, Cy5, ATTO647

The EdU cell proliferation kits are optimized and tested for these standard dyes. If you wish to purchase
another dye or azide component, please contact us for price inquiry and suggestions.

Generally, the following standard amounts of EdU are delivered with the kits:

In order to freely choose the right kit for your special in vivo application, our in vivo kits are designed in a
sense that you combine one of our three baseclick ClickTech EdU cell proliferation kits such as Imaging,
Flow Cytometry and our High-throughput Screening kit with the dye of choice and the right EdU content
for your animal model.

Depending on your animal or the number of animals to be tested you can choose between three kit sizes
S, M and L with increasing EdU content, as indicated in the following table.

Kit size

Content of EdU

S

50 mg

M

500 mg

L

1000 mg

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 EdU Cell Proliferation

in vivo Kits

4

EdU in vivo 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 analogs 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.

The baseclick EdU in vivo Kits overcome these limitations, providing a superior alternative to BrdU and
[3H]thymidine assays for directly measuring DNA synthesis in vivo. EdU (5-ethynyl-2’-deoxyuridine) is a
nucleoside analog to thymidine and is incorporated into DNA during active DNA synthesis. In contrast to
BrdU assays, the EdU in vivo Kits are not antibody based and therefore do not require DNA denaturation
for detection of the incorporated nucleoside. Instead, the EdU in vivo Kits utilize a fast, efficient and
reliable method based on the so called “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.

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.

This is not an allowance to perform in vivo studies. Contact your local authorities for the animal testing
regulations and authorization of your in vivo experiments, if not already done.

Literature Citation: When describing a procedure for publication using this product, please cite it as the
ClickTech EdU Cell Proliferation in vivo Kit from baseclick GmbH.

ClickTech EdU Cell Proliferation
in vivo Kits

5

The baseclick ClickTech EdU in vivo Kit is compatible with several cell cycle dyes and 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

Fluorescent proteins (e.g. GFP)

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

Cautions:

EdU: is a nucleoside analogue which can be incorporated into DNA. Handle and dispose EdU in compliance
with all pertaining local regulations. EdU is soluble in DMSO, water, alcohol and aqueous buffers. For in

vivo experiments, EdU can be solved in a buffered saline solution such as PBS.

EdU toxicity: No cytotoxicity could be measured up to 1 mM EdU. The data on the pharmacotoxicity of EdU

are in publication process at the moment by baseclick.

The cautions of other ingredients are described in the according User Manual you receive with your kit or
can also be downloaded from our website www.baseclick.eu.

MSDS: the appropriate MSDS for all components of each individual kit can be downloaded from our
website www.baseclick.eu.

ClickTech EdU Cell Proliferation

in vivo Kits

6

1. Material provided and storage conditions

For the specification of the material provided and their storage conditions please see the specific user
manual of your purchased kit which is included in your baseclick box or can be downloaded from our
webpage, since each kit has its proper composition.

2. Required Material and Equipment

The required materials and equipment depends on your provided kit; please see the specific kit´s user
manual delivered with the kit you purchased.

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

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

• Fixative solution (3 - 4% Paraformaldehyde in PBS)*

• Permeabilization solution (for example, 0.5% Triton® X-100 in PBS)*

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

• Deionized water or 18 MΩ purified water

* already included in flow cytometry kits

3. Preparation of the EdU stock solution

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

• For the preparation of 10 mM EdU solution, dissolve EdU in the appropriate amount of phosphate

buffered saline PBS according to Table 2 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 one year.

Table 2: Amounts of PBS needed to dissolve EdU for a final concentration of 10 mM.

EdU

Amount of PBS

50 mg

20 mL

100 mg

40 mL

500 mg

200 mL

1 g

400 mL

5 g

2000 mL

The preparations of other stock solutions are described in the according User Manual you get with your
kit or can be downloaded from our website www.baseclick.eu.

ClickTech EdU Cell Proliferation
in vivo Kits

7

4. EdU Administration

We recommend testing a range of EdU concentrations to determine the optimal concentration for your
experiment. If currently using BrdU-based assays, a similar concentration and incubation time to BrdU is a
good starting point for EdU. The optimal concentration may vary depending upon the duration of the
pulse. Lower concentrations are recommended for longer incubation time. General recommendations are
listed below (See point 5).

EdU administration to the animals can be performed following injection or media incorporation.

Successful EdU-labeling in animals has been reported for the following species:

Mouse: Salic A et al., Proc Natl Acad Sci USA 2008, 105, 2415-2420.
Rat: Guo J. et al, Cardiovascular Diabetology 2012, 11, 150.
Nematode (C. elegans): Dorsett M, Westlund B, Schedl T, Genetics 2009, 183, 233-247.
Cricket (Gryllus bimaculatus): Bando T, Mito T, Maeda Y et al., Development 2009, 136, 2235-2245.
Chicken (Gallus domesticus): Kaiser CL, Kamien AJ, Shah PA, Laryngoscope 2009, 119, 1770-1775.
Zebra Fish larva (Danio rerio): Luedtke W. et al., PNAS 2011, 108, 51.

5. General recommendations for different animals

5.1 Mouse / Rat: see page 8.

5.2 Nematode (C. elegans): see page 9.

5.3 Cricket (Gryllus bimaculatus): see page 9.

5.4 Chicken (Gallus domesticus): see page 10.

ClickTech EdU Cell Proliferation

in vivo Kits

8

5.1 Mouse / Rat:

EdU can be injected in different doses from 10 - 200 mg/kg. Using 50 mg/kg of EdU in PBS i.p. results in
near maximal intensity of proliferated cells.

Reference for mouse: Salic A et al., Proc Natl Acad Sci USA 2008, 105, 2415-2420.
Reference for rat: Guo J. et al, Cardiovascular Diabetology 2012, 11, 150.

EdU administration:

• We recommend to perform a single intraperitoneal injection of EdU in PBS (50 mg/kg).

• Euthanize the mouse 4 hours (or longer depending on the cell proliferation duration of the

tissue of interest) after EdU injection.

• Remove the desired tissue/organ.

• Snap-freeze the tissue/organ.

• Section the tissue/organ at 20 µm using a cryostat and mount onto appropriate slides.

Cell fixation and permeabilization:

For flow cytometry analysis:

• see step 6.
For imaging analysis:

• Allow slides containing mounted frozen tissue sections to thaw and warm to room

temperature.

• Fix tissue sections with 4% Paraformaldehyde in PBS (pH 7.4) for 15 minutes.

• Wash twice with 3% BSA in PBS (pH 7.4).

• Permeabilize the tissue sections with 0.5% Triton X-100 in PBS for 20 minutes.

• Wash twice with 3% BSA in PBS.

EdU detection:

• see step 7.

ClickTech EdU Cell Proliferation
in vivo Kits

9

5.2 Nematode (C. elegans):
Reference: Dorsett M, Westlund B, Schedl T, Genetics 2009, 183, 233-247.

EdU administration:

• Prepare EdU plates: grow MG1693 Escherichia coli (thymidine deficient) overnight at 37°C.

Dilute the cells 1/50 in 1% glucose, 1.25 µg/mL thiamine, 0.5 µM thymidine, 1 mM MgSO4 and
20 µM EdU in M9 minimal media. Grow this culture at 37 °C for 24 hours in the dark, harvest
bacteria and resuspend in a small volume of M9 minimal media. Plate the suspension on 60­mm M9 plates.

• For labelingproliferative cells, place the worms into the EdU plates for 3 hours.

• Wash the plates in PBS to collect the worms and dissect them.

Cell fixation and permeabilization:

For flow cytometry analysis:

• see step 6.
For imaging analysis:

• Fix sections in 3% Paraformaldehyde in 0.1 M K2HPO4 (pH 7.2) for 5 minutes at room

temperature.

EdU detection:

• see step 7.

5.3 Cricket (Gryllus bimaculatus):
Reference: Bando T, Mito T, Maeda Y et al., Development 2009, 136, 2235-2245.

EdU administration:

• We recommend to perform a single intraperitoneal injection of EdU in PBS

(50 mg/kg).

• Euthanize the cricket 4 hours after EdU injection.

• Remove the desired tissue/organ.

• Snap-freeze the tissue/organ.

• Section the tissue/organ at 20 µm using a cryostat and mount onto appropriate slides.

ClickTech EdU Cell Proliferation

in vivo Kits

10

Cell fixation and permeabilization:

For flow cytometry analysis:

• see step 6.
For imaging analysis:

• Allow slides containing mounted frozen tissue sections to thaw and warm to room

temperature.

• Fix tissue sections with 4% Paraformaldehyde in PBS (pH 7.4) for 15 minutes.

• Wash twice with 3% BSA in PBS.

• Permeabilize the tissue sections with 0.5% Triton X-100 in PBS for 20 minutes.

• Wash twice with 3% BSA in PBS.

EdU detection:

• see step 7.

5.4 Chicken (Gallus domesticus):
Reference: Kaiser CL, Kamien AJ, Shah PA, Laryngoscope 2009, 119, 1770-1775.

EdU administration:

• Perform a single subcutaneous injection of EdU (50 mg/kg) in sterile PBS (pH 7.4).

• Euthanize the bird 8 hours after EdU injection.

• Remove the tissue/organ.

• Snap-freeze the tissue/organ.

• Section the tissue/organ at 20 µm using a cryostat and mount onto appropriate slides.

Cell fixation and permeabilization:

For flow cytometry analysis:

• see step 6.
For imaging analysis:

• Fix the cells in chilled 4% Paraformaldehyde in PBS for 1 hour.

• Rinse 3 times in PBS for 5 minutes each rinse.

EdU detection:

• see step 7.

ClickTech EdU Cell Proliferation
in vivo Kits

11

6. Cell fixation and permeabilization for flow cytometry analysis

See also according User Manual

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

6.1 Wash the cells with 3 mL of 1% BSA in PBS. Pellet the cells and remove the supernatant.

6.2 Dislodge the cell pellet. Add 100 µL of the fixative solution to the cells. Mix well and incubate for

15 minutes at room temperature. Protect from light.

6.3 Remove the fixative solution and wash the cells with 3 mL of 1% BSA in PBS. Pellet cells and

remove the supernatant. Remove all residual blood cell debris and haemoglobin before
proceeding.

6.4 Dislodge the cell pellet. Resuspend the cells in 100 µL of 1x saponin-based permeabilization buffer

in PBS. Mix well and proceed to step 7 for the click reaction.

7. EdU detection

See according User Manuals

8. Imaging/flow cytometry analysis

See according User Manuals

ClickTech EdU Cell Proliferation

in vivo Kits

12

9. Example of the data derived from an EdU in vivo Kit based experiment:

This EdU in vivo kit allows detection of cell proliferation in whole animals, showing no toxic effects. Cell
proliferation is detected in different organs by applying the reagents of this kit. In healthy mice cell
proliferation is nicely detected in lymphnodes, but not in other organs showing the reliability of this
baseclick method.

Figure 1: NMRI mice treated with different concentrations of EdU intraperitoneally and sacrificed after 4 hrs. A flow cytometric
method is used to analyze the distribution of EdU in different organs.

The shown in vivo experiments were performed in collaboration with a third party after having received
the allowance of the local authorities.