Multiplex Cell Fate Tracking by Flow Cytometry - MDPI
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Protocol
Multiplex Cell Fate Tracking by Flow Cytometry
Marta Rodríguez-Martínez 1, *, Stephanie A. Hills 2 , John F. X. Diffley 2 and Jesper Q. Svejstrup 1, *
1 Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
2 Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
* Correspondence: marta.rodriguez-martinez@crick.ac.uk (M.R.-M.); jesper.svejstrup@crick.ac.uk (J.Q.S.)
Received: 18 June 2020; Accepted: 15 July 2020; Published: 17 July 2020
Abstract: Measuring differences in cell cycle progression is often essential to understand cell behavior
under different conditions, treatments and environmental changes. Cell synchronization is widely
used for this purpose, but unfortunately, there are many cases where synchronization is not an option.
Many cell lines, patient samples or primary cells cannot be synchronized, and most synchronization
methods involve exposing the cells to stress, which makes the method incompatible with the study
of stress responses such as DNA damage. The use of dual-pulse labelling using EdU and BrdU can
potentially overcome these problems, but the need for individual sample processing may introduce a
great variability in the results and their interpretation. Here, we describe a method to analyze cell
proliferation and cell cycle progression by double staining with thymidine analogues in combination
with fluorescent cell barcoding, which allows one to multiplex the study and reduces the variability
due to individual sample staining, reducing also the cost of the experiment.
Keywords: BrdU; EdU; fluorescent cell barcoding
1. Introduction
Assessing cell proliferation and cell cycle distribution is often at the basis of cell behavior studies.
One of the most widely used methods to study cell proliferation, and in particular cell cycle progression
under different conditions, is flow cytometry. Since it was first used to study cell cycle distribution [1],
several methods have been developed to allow the analysis of cell proliferation [2]. The simplest
approach is to measure DNA content across the cell population at a single time point [3], alone or in
combination with other cell cycle markers, such as cyclins [4–6]. However, this “snapshot” method
provides limited information about cell cycle kinetics. To overcome this problem, methods based on
cell synchronization and release have been widely used [7,8]. Unfortunately, two main problems are
encountered when using these techniques: (i) many of these methods rely on chemical or biological
agents to achieve synchronization, most of which are stress-inducing, which makes them incompatible
with the study of cellular responses to other cellular stresses (such as DNA damage), and (ii) many cell
types, such as patient cells, primary cells or several transformed cell lines cannot be synchronized.
There is a third approach that partially overcomes this problem: the incorporation of thymidine
analogues in a time-lapse manner [9]. This allows an estimation of the progression rate through
different phases of the cell cycle. To this end, BrdU and other analogues have been widely used [10–16];
most recently, EdU [16–19] and click chemistry, which does not require denaturation of the sample
before analysis. However, since only one nucleotide analogue can be used, this technique requires the
acquisition of individual samples at each time point of interest, which is not always possible when the
amount of sample is limited (i.e., in vivo studies or patient samples). Moreover, the power of single
labelling is limited as it can only assess the number of cells in each cell cycle phase at a given time
point to infer cell cycle dynamics, while it cannot follow the evolution of specific populations (i.e., it
cannot be used to detect from where a certain cell population arose, or where it progressed to, in the
Methods Protoc. 2020, 3, 50; doi:10.3390/mps3030050 www.mdpi.com/journal/mpsMethods Protoc. 2020, 3, 50 2 of 9
time window of study, for example, after a certain treatment). The problems encountered in single
labelling can be partially solved by the use of double labelling with EdU and BrdU [20]. However, the
necessity to individually stain and analyze samples often introduces a bias in the results that can easily
lead to misinterpretation of the data. For example, when studying cell cycle distribution, differences in
cell number or dye concentration may result in changes in the position of the G1-phase peak, making it
difficult to differentiate these variations from biologically relevant ones.
Here, we describe a protocol to combine the use of BrdU and EdU in combination with fluorescent
cell barcoding (FCB) [21,22] and DNA dyes. FCB offers a solution to the variability in sample staining
and analysis encountered when using the methods listed above. First, it allows for a multiplexed flow
cytometry analysis and introduces a normalization of samples which ensures unbiased comparison
and detection of small differences. Second, this approach also reduces the amount of antibody and
staining solutions needed, resulting in a high reduction of costs. This method can help overcome some
restrictions of the currently available methods for analysis of the cell cycle and cell proliferation.
2. Experimental Design
An overview of the procedure is shown in Figure 1. This method has been optimized for Flp-In
T-REx 293 cells growing in monolayer, but it can be extended to other mammalian cell types. First,
cells are labelled with BrdU and EdU following the user’s experimental setup. For example, different
time points, cell lines and treatments can be combined to compare the desired parameters in a specific
experiment. In this case, the monolayer nature of the culture allows the first analogue to be washed off
before labelling with the second analogue, but different approaches can be considered. After harvesting,
cells are washed, fixed, permeabilized and denatured, to allow BrdU antibody detection. Then, cells
are stained with barcode dyes, allowing the desired populations to be combined and compared. Lastly,
unbiased cell staining of the combined population is performed using BrdU antibodies, EdU click-iT
chemistry and a DNA dye. The study presented here has been optimized for DAPI staining, but other
dyes may be considered.
The success of this method is dependent on the efficient cellular uptake of the thymidine analogue
and efficient labelling of newly synthesized DNA. It is therefore critical to check the efficiency of BrdU
and EdU incorporation in the specific cell type and experimental setup. It is also advised to check the
fluorescent cell barcoding efficiency in the chosen system. A control for the cross-reactivity of the BrdU
vs. EdU detection is recommended. Ideally, the control should include two samples, labelled with
EdU or BrdU respectively, and combined by FCB to perform the staining procedure on both samples
simultaneously. This would allow any cross-reactivity between BrdU and EdU detection methods to
be assessed as well as verification of the correct separation of cell populations by FCB. Controls for
individual labelling should be included to help define flow cytometry analyzer parameters, as well as
compensation, if required.
Interestingly, FCB has been shown to be compatible with fixable viability dyes [22], despite the
common nature of the modified molecule, as both are based on amine esterification. This is not included
in this protocol, but the use of such dyes may be implemented, if required. The main limitation of
the method is that the denaturation step necessary for BrdU staining is not compatible with regular
antibody staining.
A scheme summarizing the experimental design, including the time needed to complete every
stage, is provided in Figure 1.Methods Protoc. 2020, 3, 50 3 of 9
Condition 1 Condition 2 Condition 3 TIME
BrdU pulse User
EdU pulse dependent
Harvest 15 min
Fixation 15 min
Permeabilization 20 min
Denaturation 30 min
FCB 10 min
washes
10 min
Pool
Antibody staining 2 hours
Click-it 1 hour
DAPI + RNAse A 20 min
ANALYSIS
Figure 1. Schematic representation of the method.
2.1. Materials
Figure
• 1
Flp-In T-Rex 293 Cell Line (Thermo Fisher Scientific, Gloucester, UK; Cat.no.: R78007,
RRID:CVCL_U427, authenticated by Thermo Fisher Scientific and routinely confirmed to be
mycoplasma-free), or another mammalian cell line or system of choice.
• High glucose DMEM–Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Gloucester,
UK; Cat.no.: 11965118).
• Gibco Fetal Bovine Serum (Thermo Fisher Scientific, Gloucester, UK; Cat.no.: 10270098).
• PBS (Phosphate-buffered saline, VWR, Poole, UK; Cat.no.: 45000).
• Trypsin-EDTA Solution 0.25% (Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: T4049.
• Formaldehyde solution 37% (Sigma-Aldrich LTD, UK; Cat.no.: F1635).
• Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific,
Gloucester, UK; Cat.no.: C10634). A different Alexa dye should not affect the results of the protocol.
• BrdU (5-Bromo-20 -Deoxyuridine, Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: B5002-1G).
• BrdU Monoclonal Antibody (MoBU-1) (Thermo Fisher Scientific, Gloucester, UK; Cat.no.: B35141,
RRID:AB_2536441).
• For FBC. Alexa Fluor 488 NHS Ester (Succinimidyl Ester) (Thermo Fisher Scientific, Gloucester,
UK; Cat.no.: A20000). A different Alexa dye should not affect the results of the protocol.
• Hydrochloric acid 37% (Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: 258148).
• Bovine serum albumin (BSA) (Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: A3983).Methods Protoc. 2020, 3, 50 4 of 9
• Ethanol absolute 99.8+% (Thermo Fisher Scientific, Gloucester, UK; Cat.no.: 10437341).
• Goat Anti-Mouse IgG H&L (Alexa Fluor 555) (Abcam, Cambridge, UK; Cat.no.: ab150114,
MethodsRRID:AB_2687594). A different
Protoc. 2020, 3, x FOR PEER REVIEW Alexa dye should not affect the results of the protocol. 5 of 9
• 0
DAPI (4 ,6-diamidino-2-phenylindole, Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: D9542).
• 6 RNase
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staining, remove any
we recommend non-incorporated
using onlydye before
3 dilutions, pooling
as indicated the samples.
in Table 1.
4.4.
4. Pool
samples, spin
Pool samples,
samples, Table
spin down
spin
down and
2. down
Parameters
and remove
remove supernatant.
and remove
used in the
supernatant.
supernatant.
flow cytometry analyzer LSRII.
1.
5. Each sampleSTEP:
PAUSE will be stainedcan
Samples with be one
kept concentration
O/N at 4◦°Cof the dye. To do so, add 3 µL diluted dye
5.5. Table
PAUSE STEP:
PAUSE 1. Serial
STEP: Samples dilution
Samplescan to be
canbe done
bekept from
keptO/N O/Nthe in 1in
inmg/mL
at44 °C
at CNHS
wash
wash
wash
buffer.
Ester stock solution.
buffer.
buffer.
Reagent Laser
to 147 µL wash buffer (70% ethanol can also be used). For the compensation Bandpass Filter control, the highest
3.4. BrdU Antibody Staining
concentration Final
FCB (Alexa
is recommended. 488) 488
and EdU Click-iT. Time for Completion: 03:00 h Dye 525/50
3.4. BrdU
3.4. BrdU Antibody
Antibody Staining Staining and and EdUEdU Make 50x Time
Click-iT.
Click-iT. Timefor forCompletion:
Completion:03:00 03:00hh DMSO
2. Add the total 150BrdU
Concentration µL(Alexa
to the555) sample and561 incubate(fromfor 10 min 582/15
Previous Dilution)
at RT.
1. CRITICAL STEP Add 200 µL 1:50 BrdU Monoclonal Antibody (MoBU-1) in wash buffer to
1.
1.3. CRITICALµg/ml
CRITICAL
CRITICAL STEP Add
STEP
STEP Add 200
Wash 200
3 ×µL µL
µg/ml 1:50
1501:50 BrdUMonoclonal
µL BrdU
wash Monoclonal
buffer. It isµL Antibody
very
Antibodyimportant (MoBU-1) µLinin wash
to thoroughly
(MoBU-1) wash buffer
bufferthe
wash to
to
each sample and incubate 45 min at RT. It is absolutely necessary to use this specific antibody
each sample
samples
each sample and
to remove
and incubate
15incubate 45 minmin
any non-incorporated
45 at RT.
750at RT. ItItdyeisisabsolutely
absolutely
before pooling 75 necessary
necessary to use
the samples.
to use this
this specificantibody
25specific antibody
clone (MoBU-1), as it has no cross reactivity with EdU.
clone
4. clone (MoBU-1),
Pool samples,
(MoBU-1), spin5as it
as down has
it has no no cross
andcross 250
remove reactivity
supernatant.
reactivity with
with EdU. EdU. 33.3 66.6
2. Wash 3 × 150 µL wash buffer.
2.2.
5. WashWash PAUSE
33 ×× 150
150 µL1.3
STEP:
µL wash
Samples
wash buffer.
buffer. can be 65kept O/N in at 4 °C wash
3. Add 200 µL 1:200 Goat Anti-Mouse Alexa Fluor (555) in wash buffer to each sample and incubate
26 buffer. 74
3.
3. Add
Add 200
200atµL µL 1:200 0.3 Goat Anti-Mouse 15 Alexa Fluor (555)
1:200 Goat Anti-Mouse Alexa Fluor (555) in wash buffer to each sample and in wash
23.1 buffer to each sample
76.9 andincubate
incubate
45 min RT.
45
3.4. BrdU min at
Antibody RT. Staining
0.075 and EdU Click-iT.
3.75 Time for Completion: 25 03:00 h 75
4. 45 Washmin3at× RT. 150 µL wash buffer.
4. Wash 3 × 150 µL0wash buffer. 0 0 100
4.1.
5. WashPerform 3 ×Click-iT
CRITICAL 150 µLSTEP wash
reaction buffer.
Add 200 µL 1:50
following theBrdU Monoclonalinstructions.
manufacturer’s Antibody (MoBU-1) in wash buffer to
5. The Perform
three Click-iT
recommended reaction following
dilutions to the together
use manufacturer’s in the instructions.
same experiment after
5. Perform
each sample Click-iT and reaction
incubatefollowing
45 min atthe RT.manufacturer’s
It is absolutelyinstructions. necessary to use thisdenaturing are
specific antibody
indicated
3.5. RNase
clone A in either
Treatmentasand
(MoBU-1), dark or
DAPI
it has light green
noStaining. (i.e., 15, 1.3
Time forwith
cross reactivity and 0.075
Completion: or
EdU. 00:20 5, 0.3 andh 0).
3.5. RNase A Treatment and DAPI Staining. Time for Completion: 00:20 h
3.5.
2. RNase
Wash A3 Treatment
× 150 µL and DAPI
wash buffer. Staining. Time for Completion: 00:20 h
1. Each
1. Resuspend
samplecells willin be200 µL wash
stained withbuffer containing 100
one concentration ofµg/mL
the dye. RNase
To doA and
so, add 1 µg/mL DAPI and
3 µL diluted dye
1. Resuspend cells inGoat
200 µL wash buffer containing 100 µg/mL RNase Aeach
and 1 µg/mL DAPI and
1.3. Resuspend
Add 200 µL
incubate
to 147 µLfor for
wash
1:200
cells
15 in
min. 200 Anti-Mouse
µL wash Alexa
buffer Fluor (555)
containing 100 inµg/mL
wash buffer
buffer (70% ethanol can also be used). For the compensation control, the highest RNase toA and sample
1 µg/mL and incubate
DAPI and
incubate
45 min atfor RT. 15 min.
2. incubate
Centrifuge
concentration 15ismin.
and resuspend
recommended. in 200 µL wash buffer.
2.
4. Centrifuge
Wash 3 × and
150 µL resuspend
wash buffer.in 200 µL wash buffer.
2.2.
3. Centrifuge
AddCRITICAL
the total and150 resuspend
STEP µL to theinsample
Transfer 200 µLand
solution wash to buffer.
a 5 mLfor
incubate round
10 min bottom
at RT.polystyrene test tube with cell
3. CRITICAL STEP
5. Perform Click-iT reaction following Transfer solution the to a 5 mL round
manufacturer’s bottom
instructions. polystyrene test tube with cell
strainer
3.3. strainerCRITICAL cap. It is necessary
STEP to
Washto3 filterfilter
×solution
150thethe cell
µL cell
wash suspension
5buffer. through
It through
is very the tube
important cap to avoid
to thoroughly cellwith
clumps.
CRITICAL cap. It is STEP Transfer
necessary to asuspension
mL round bottom thepolystyrene
tube cap totest
avoid cellwash
tube the
cell
clumps.
samples
strainer
3.5. RNase A cap. to remove
Treatment any
It is necessary non-incorporated
and DAPItoStaining. filter theTime dye
cell suspension before
for Completion: pooling
through the
00:20the samples.
h tube cap to avoid cell clumps.
3.6.
4. Analyze in Flow spin
Pool samples, Cytometrydown Analyzer.
and remove Time for Completion: Defined by Experimental Design
supernatant.
3.6. Analyze in Flow Cytometry Analyzer. Time for Completion: Defined by Experimental Design
1. Resuspend cells inSamples
200 µL wash buffer
kept containing 100 µg/mL RNase A and 1 µg/mL DAPI and
●
5. The PAUSE
parameters STEP: used in thiscan be
illustrative O/Nexample in at 4are °Cshownwash buffer.
in Table 2; however, others can be
● The parameters
incubate for 15 min. used in this illustrative example are shown in Table 2; however, others can be
used depending on the user requirements and availability.
2. used
3.4. BrdU depending
Centrifuge
Antibody andStaining on the
resuspend user
and EdU requirements
in 200 µL wash
Click-iT. Timeand
buffer. availability.
for Completion: 03:00 h
3. CRITICAL STEP Transfer solution to a 5 mL round bottom polystyrene test tube with cell
1. strainer CRITICAL Table 2. Parameters used in the flowMonoclonal
cytometry analyzer LSRII.
It isSTEP
cap.Table necessaryAdd 200
2. Parameters µL 1:50
to filter
used the BrdU
cell
in the suspension
flow cytometrythrough Antibody
analyzer the tube
LSRII. (MoBU-1) in wash
cap to avoid cellbuffer
clumps. to
each sample and incubate 45 min at RT. It is absolutely necessary to use this specific antibodyMethods Protoc. 2020, 3, 50 6 of 9
3.6. Analyze in Flow Cytometry Analyzer. Time for Completion: Defined by Experimental Design
• The parameters used in this illustrative example are shown in Table 2; however, others can be
used depending on the user requirements and availability.
Table 2. Parameters used in the flow cytometry analyzer LSRII.
Reagent Laser Bandpass Filter
FCB (Alexa 488) 488 525/50
BrdU (Alexa 555) 561 582/15
EdU (Alexa 647) 633 660/20
DAPI (UV) 355 450/50
4. Expected Results
The analysis of the samples should show a good separation of the pooled samples by the FCB
parameter. It should also show no cross reactivity between BrdU and EdU.
Figure 2 provides a description of the analysis and results to expect in a successful experiment.
Condition 1 Condition 2 Condition 3
BrdU pulse EdU pulse BrdU pulse
EdU pulse
ANALYSIS
Initial population doublet/debris After doublet/debris
exclusion method exclusion
250K
10 5 10 5
Condition 1
DAPI (H)
200K
104 104
FCB
FCB
150K Condition 2
103 100K 103
Condition 3
0 50K
0
-103 -103
0
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
DAPI (A) DAPI (A) DAPI (A)
Condition 1 Condition 2 Condition 3
105 posibility 1 posibility 2
104
EdU
103
0
-103
105
104
BrdU
103
0
-103
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
DAPI DAPI DAPI DAPI
Figure 2. Expected results for the experiment. In this example, three different conditions are used for
labelling the cells as indicated by conditions 1 to 3. The analysis performed in FlowJo should look
as shown in theFigure 2
figure. Firstly, the populations can be easily distinguished by FCB, even before cell
doublets and debris exclusion. Afterwards, gating is used to separate these populations and analyze
BrdU and EdU staining. As expected, no cross reactivity is observed between BrdU and EdU (condition
1 and 2). Examples of two possible outcomes are shown for condition 3. In these, the variability in BrdU
and BrdU staining are exemplified (note that possibility 2 is from a different experiment). Importantly,
this variability does not affect the interpretation of the experiment, as the use of FBC (allowing all
samples to be stained in the same tube) ensures that any observed changes are not due to variations in
staining. This, and other potential issues arising, and their troubleshooting, are discussed in Table 3.
Colors in the flow analysis graphs indicate cell population density, as defined by FlowJo analysis tools.Methods Protoc. 2020, 3, 50 7 of 9
Table 3. Potential issues arising and respective troubleshooting.
Issue Possible Causes Suggestions
Cell loss Cell loss during centrifugation Use swing rotor as recommended
1. Pipette up and down to ensure
1. Cells had formed aggregates
cell dispersion after 3.2
after step 3.2
2. Do not exceed 4 × 106 cells per
2. Too many cells used
Inefficient FCB detection FCB dilution (12 × 106
for labelling
cells total)
3. Not enough washing before
3. Increase wash volume
pooling the samples after FCB
and time
1. Dye is too old
Insufficient FCB population 1. Use freshly prepared dilutions
2. Cell type used requires
separation 2. Optimize dilutions prior to use
different dilutions
1. Increase washing time
1. Not enough washes after 3.2
and volume
Poor BrdU detection 2. Too many cells used
2. Do not exceed 12 × 106
for labeling
cells total
1. Increase washing time
1. Not enough washes after 3.2
and volume
Poor EdU detection 2. Too many cells used
2. Do not exceed 12 × 106
for labeling
cells total
5. Reagents Setup
5.1. Tissue Culture Medium
• High glucose DMEM
• 10% v/v FBS
• 100 U/mL penicillin
• 100 µg/mL streptomycin
5.2. Wash buffer
• PBS
• 1% BSA
5.3. NHS Ester Stock Solution
Prepare 1 mg/mL in DMSO
5.4. BrdU 100µM
Prepare 10 mM stock (1:1000) in DMSO
Author Contributions: Conceptualization, M.R.-M.; methodology, M.R.-M. and S.A.H.; validation, M.R.-M. and
S.A.H.; writing—original draft preparation, M.R.-M.; writing—review and editing, M.R.-M.,S.A.H. and J.Q.S.;
supervision, J.F.X.D. and J.Q.S.; funding acquisition, J.F.X.D. and J.Q.S. All authors have read and agreed to the
published version of the manuscript.Methods Protoc. 2020, 3, 50 8 of 9
Funding: This work was supported by the Francis Crick Institute (FCI), which receives its core funding from
Cancer Research UK (FC001166), the UK Medical Research Council (FC001166), and the Wellcome Trust (FC001166),
and by a grant from the European Research Council, Agreements 693327 (TRANSDAM) to JQS.
Acknowledgments: We thank the flow cytometry facility of the Francis Crick Institute for their help and support,
and especially Derek Davies for his ideas and input for this manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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