Protocol Multiplex Cell Fate Tracking by

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: [email protected] (M.R.-M.); [email protected] (J.Q.S.)

 Received: 18 June 2020; Accepted: 15 July 2020; Published: 17 July 2020 

Abstract: Measuring differences in 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 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/mps Methods 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 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 Figure1. 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 , 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 Figure1. 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 Flp-In T-Rex 293 Cell Line (Thermo Fisher Scientific, Gloucester, UK; Cat.no.: R78007, Figure• 1 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-2 -Deoxyuridine, Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: B5002-1G). • 0 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). Protoc. 2020, 3, x FOR PEER A diREVIEWfferent Alexa dye should not affect the results of the protocol. 5 of 9 DAPI (4 ,6-diamidino-2-phenylindole, Sigma-Aldrich LTD, Gillingham, UK; Cat.no.: D9542). • 0 6 RNasedilutions A, DNasein non-denaturing and protease-free conditions. (10 mg /However,mL) (Thermo after Fisher the denaturation Scientific, Gloucester, step necessary UK; Cat.no.: for • BrdUEN0531). staining, we recommend using only 3 dilutions, as indicated in Table 1.

2.2. EquipmentTable 1. Serial dilution to be done from the 1 mg/mL NHS Ester stock solution. Biological safetyFinal cabinet type II Dye • Make 50x DMSO CO2 incubatorConcentration (from Previous Dilution) • Chemical fumeµg/ml hood µg/ml µL µL • Tissue culture15 plates of the desired750 size (for instance, Corning,75 Deeside, UK;25 Cat.no.: 3506) • Low binding/maximum5 recovery250 1.5 mL microcentrifuge33.3 tubes (for instance,66.6 Axigen, Corning, • Deeside, UK; Cat.no.:1.3 11311984)65 26 74 Swing rotor centrifuge0.3 for 1.5 mL15 microcentrifuge tubes23.1 (for instance, Eppendorf,76.9 Stevenage, UK; • Cat.no.: 5804R0.075 with 1.5 ml adapters)3.75 25 75 Falcon 5 mL Round0 Bottom Polystyrene0 Test Tube with0 Cell Strainer Cap100 (Falcon, Corning, • TheDeeside, three recommended UK, Cat.no.: 352235)dilutions to use together in the same experiment after denaturing are Methods Protoc. 2020, 3, x FOR PEER REVIEW 5 of 9 indicatedRotating in wheel either fordark 1.5 or mLlight microcentrifuge green (i.e., 15, 1.3 tubes and 0.075 (for instance,or 5, 0.3 and SB3, 0). Cole-Parmer, Saint Neots, UK; • Cat.no.: 11496548) 1. Each6 dilutions sample willin non-denaturing be stained with conditions. one concentrat However,ion of theafter dye. the To denaturation do so, add 3step µL dilutednecessary dye for Flow cytometry analyzer (for instance, LSR II, BD Biosciences, UK) • to BrdU147 µL staining, wash buffer we recommend (70% ethanol using can onlyalso be3 dilutions, used). For as the indicated compensation in Table control, 1. the highest concentration is recommended. 3. Procedure 2. Add theTable total 1. 150 Serial µL dilution to the sampleto be done and from incubate the 1 mg/mL for 10 NHSmin atEster RT. stock solution. 3. AllCRITICAL centrifugations STEPFinal Wash should 3 be× 150 performed µL wash in buffer. a swing It rotoris veDyery centrifuge important at roomto thoroughly temperature wash (RT) the to Make 50x DMSO minimizesamples to cell removeConcentration loss. any non-incorporated dye(from before Previous pooling theDilution) samples. 4. PoolIt samples, is critical spin toµg/ml pipette down up and and removeµg/ml down supernatant. after every step to µL avoid cell aggregation. µL 5. PAUSE STEP:15 Samples can be750 kept O/N in at 4 °C wash75 buffer. 25 All centrifugations are performed at 300 g and RT, 3 min unless stated otherwise. 5 250 33.3 66.6 3.4. BrdU Antibody Staining and EdU Click-iT. Time for Completion: 03:00 h 3.1. Cell EdU and BrdU1.3 Labelling. Time for65 Completion: Defined by26 Experimental Design.74 1 Day in This Setup 1. CRITICAL STEP0.3 Add 200 µL 1:5015 BrdU Monoclonal23.1 Anti body (MoBU-1)76.9 in wash buffer to 1. Seed Flp-In T-REx 293 cells in 6 well plates at a density of 5 105 cells/well. Add 3 mL of tissue each sample and0.075 incubate 45 min3.75 at RT. It is absolutely necessary25 × to use this specific75 antibody culture medium and leave overnight (O/N) in a CO incubator at 37 C and 5% CO . clone (MoBU-1), as0 it has no cross reactivity0 with EdU.2 0 ◦ 100 2 2. Add EdU and BrdU as required by the experiment. A 30 min 10 µM pulse of each analogue is 2. WashThe 3three × 150 recommended µL wash buffer. dilutions to use together in the same experiment after denaturing are commonly used for cell cycle studies. 3. Addindicated 200 µL in 1:200 either Goat dark Anti-Mouse or light green Alexa (i.e., 15, Fluor 1.3 and (555 0.075) in wash or 5, 0.3 buffer and 0).to each sample and incubate 45 min at RT. 3.2. Harvest, Fixation, Permeabilization and Denaturation. Time for Completion: 01:20 h 4. 1. WashEach 3 sample× 150 µL will wash be stainedbuffer. with one concentration of the dye. To do so, add 3 µL diluted dye 5.1. PerformHarvestto 147 µLClick-iT cells wash by reactionbuffer washing (70% following once ethanol with the PBScan manufacturer’s andalso addingbe used). 250 instructions.ForµL the trypsin compensation to a well ofcontrol, a 6-well the plate highest for 3concentration min or until cellsis recommended. are fully detached (other harvesting methods may be used, depending on the 3.5.2. RNasecellAdd A type the Treatment total and experiment150 and µL DAPI to the setup).Stai samplening. Neutralize Timeand incubate for Completion: trypsin for 10 by min00:20 adding at h RT. 1 mL of tissue culture medium and transfer cells to a 1.5 mL low binding tube. Wash once with PBS. 1. 3. Resuspend CRITICAL cells in STEP 200 µLWash wash 3 × buffer 150 µL contai washning buffer. 100 It µg/mL is very RNase important A and to 1thoroughly µg/mL DAPI wash and the 2. Resuspend in 500 µL of freshly prepared 4% formaldehyde and incubate 10 min at RT in the dark. incubatesamples for to 15 remove min. any non-incorporated dye before pooling the samples. Wash once with 500 µL wash buffer. 2. 4. CentrifugePool samples, and resuspend spin down in and 200 remove µL wash supernatant. buffer.

3. 5. CRITICAL PAUSEPAUSE STEPSTEP STEP:: AfterSamples Transfer wash, can solution the be mixkept to can O/N a 5 be mLin stored at round 4 °C at wash 4bo◦Cttomfor buffer. uppolystyrene to one week test in tube wash with bu ffceller. 3. Resuspend in 500 µL 70% ethanol and incubate 20 min at 20 C. Wash once with 500 µL strainer cap. It is necessary to filter the cell suspension through the− tube◦ cap to avoid cell clumps. 3.4. BrdUwash Antibody buffer. Staining and EdU Click-iT. Time for Completion: 03:00 h 3.6.4.1. AnalyzeResuspend CRITICAL in Flow in Cytometry 500 STEPµL 2NAdd Analyzer. HCl 200 and µL Time incubate1:50 for BrdU Completion: 20 Monoclonal min at RT.Defined Wash Anti bybodytwice Experimental (MoBU-1) with 500 Designµ inL wash buffer buffer. to ● Theeach parameters sample and used incubate in this 45illustrative min at RT. exampl It is abe aresolutely shown necessary in Table to2; usehowever, this specific others antibodycan be usedclone depending (MoBU-1), on as the it hasuser no requirements cross reactivity and withavailability. EdU. 2. Wash 3 × 150 µL wash buffer. 3. Add 200 µL 1:200 Goat Anti-Mouse Alexa Fluor (555) in wash buffer to each sample and incubate Table 2. Parameters used in the flow cytometry analyzer LSRII. 45 min at RT. 4. Wash 3 × 150 µL Reagentwash buffer. Laser Bandpass Filter 5. Perform Click-iTFCB reaction(Alexa 488) following the 488 manufacturer’s instructions. 525/50 BrdU (Alexa 555) 561 582/15 3.5. RNase A Treatment and DAPI Staining. Time for Completion: 00:20 h

1. Resuspend cells in 200 µL wash buffer containing 100 µg/mL RNase A and 1 µg/mL DAPI and incubate for 15 min. 2. Centrifuge and resuspend in 200 µL wash buffer. 3. CRITICAL STEP Transfer solution to a 5 mL round bottom polystyrene test tube with cell strainer cap. It is necessary to filter the cell suspension through the tube cap to avoid cell clumps.

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

Methods Protoc. 2020, 3, x FOR PEER REVIEW 5 of 9

6 dilutions in non-denaturing conditions. However, after the denaturation step necessary for BrdU staining, we recommend using only 3 dilutions, as indicated in Table 1.

Table 1. Serial dilution to be done from the 1 mg/mL NHS Ester stock solution.

Final Dye Make 50x DMSO Concentration (from Previous Dilution) µg/ml µg/ml µL µL 15 750 75 25 5 250 33.3 66.6 1.3 65 26 74 0.3 15 23.1 76.9 0.075 3.75 25 75 0 0 0 100 The three recommended dilutions to use together in the same experiment after denaturing are indicated in either dark or light green (i.e., 15, 1.3 and 0.075 or 5, 0.3 and 0).

Methods1. Each Protoc. sample2020, 3 ,will 50 be stained with one concentration of the dye. To do so, add 3 µL diluted5 dye of 9 to 147 µL wash buffer (70% ethanol can also be used). For the compensation control, the highest concentration is recommended. 3.3. Fluorescent Cell Barcoding (FCB). Time for Completion: 00:20 h 2. Add the total 150 µL to the sample and incubate for 10 min at RT. 3. CRITICAL STEP STEP WashUse the 3 × previously150 µL wash prepared buffer. It NHS is very Ester important stock solutionto thoroughly to prepare wash the dilutionssamples indicated to remove in Table any1. non-incorporated Dilutions can be kept dye atbefore20 poolingC for a fewthe weeks.samples. It is important to verify − ◦ how4. Pool many samples, dilutions spin can down be used and effi removeciently supernatant. in the cell type of choice. We have used up to 6 dilutions in5.Methods non-denaturing Protoc. PAUSE 2020 STEP, 3, conditions.x FOR: Samples PEER REVIEW However, can be kept after O/N the in denaturation at 4 °C wash step buffer. necessary for BrdU staining,5 weof 9 recommend using only 3 dilutions, as indicated in Table1. 3.4. BrdU6 dilutions Antibody in Staining non-denaturing and EdU Cconditions.lick-iT. Time However, for Completion: after the03:00 denaturation h step necessary for BrdU staining,Table 1.weSerial recommend dilution to using be done only from 3 dilutions, the 1 mg/mL as indicated NHS Ester in stock Table solution. 1. 1. CRITICAL STEP Add 200 µL 1:50 BrdU Monoclonal Antibody (MoBU-1) in wash buffer to each sample and incubate 45 min at RT. It is absolutelyDye necessary to use this specific antibody FinalTable Concentration 1. Serial dilution to Make be done 50x from the 1 mg/mL NHS Ester stock solution. DMSO clone (MoBU-1), as it has no cross reactivity with(from EdU. Previous Dilution) Final Dye 2. Wash 3µ ×g 150/ml µL wash buffer.Makeµ g/ml 50x µL DMSO µL Concentration (from Previous Dilution) 3. Add 200 µL15 1:200 Goat Anti-Mouse750 Alexa Fluor (555) in wash75 buffer to each sample 25and incubate 45 min at RT. µg/ml µg/ml µL µL 5 250 33.3 66.6 4. Wash 3 × 150 µL15 wash buffer. 750 75 25 1.3 65 26 74 5. Perform Click-iT5 reaction following250 the manufacturer’s33.3 instructions. 66.6 0.3 1.3 1565 2623.1 74 76.9 3.5. RNase A 0.075Treatment0.3 and DAPI Stai3.75ning.15 Time for Completion:23.125 00:20 h 76.9 75 0.075 3.75 25 75 1. Resuspend0 cells in 200 µL wash0 buffer containing 100 µg/mL0 RNase A and 1 µg/mL100 DAPI and 0 0 0 100 Theincubate three recommended for 15 min. dilutions to use together in the same experiment after denaturing are indicated in either 2. darkCentrifugeThe orthree light greenrecommended and (i.e.,resuspend 15, 1.3 dilutions and in 0.075 200 orµLto 5, use 0.3wash andtogether buffer. 0). in the same experiment after denaturing are indicated in either dark or light green (i.e., 15, 1.3 and 0.075 or 5, 0.3 and 0). 3. CRITICAL STEP Transfer solution to a 5 mL round bottom polystyrene test tube with cell µ 1. strainerEach sample cap. It will is necessary be stained to with filter one the concentration cell suspension of through the dye. the To dotube so, cap add to 3 avoidL diluted cell clumps. dye to 1. 147Eachµ Lsample wash buwillff erbe (70% stained ethanol with canone alsoconcentrat be used).ion Forof the the dye. compensation To do so, add control, 3 µL thediluted highest dye to 147 µL wash buffer (70% ethanol can also be used). For the compensation control, the highest 3.6. Analyzeconcentration in Flow isCytometry recommended. Analyzer. Time for Completion: Defined by Experimental Design 2. Addconcentration the total 150is recommended.µL to the sample and incubate for 10 min at RT. ●2. TheAdd parameters the total 150 used µL toin thethis sample illustrative and incubateexample forare 10 shown min at in RT. Table 2; however, others can be 3. CRITICAL STEP Wash 3 150 µL wash buffer. It is very important to thoroughly wash the 3. used CRITICAL depending STEP on the Wash user 3requirements× × 150 µL wash and buffer. availability. It is ve ry important to thoroughly wash the samples to remove any non-incorporated dye before pooling the samples. samples to remove any non-incorporated dye before pooling the samples. 4.4. Pool samples,Table spin 2. downdownParameters andand removeremoveused in the supernatant.supernatant. flow cytometry analyzer LSRII. 5. PAUSEPAUSE STEPSTEP: Samples can be kept O/N in at 4 °C wash buffer. 5. : SamplesReagent can be keptLase O/Nr in at 4 ◦C washBandpass buffer. Filter FCB (Alexa 488) 488 525/50 3.4.3.4. BrdUBrdU AntibodyAntibody StainingStaining andand EdUEdU Click-iT.Click-iT. TimeTime for for Completion: Completion: 03:0003:00 hh BrdU (Alexa 555) 561 582/15 1.1. CRITICALCRITICAL STEPSTEP AddAdd 200200 µµLL 1:501:50 BrdUBrdU MonoclonalMonoclonal AntibodyAntibody (MoBU-1)(MoBU-1) inin washwash bubufferffer toto

each sample and incubate 45 min at RT.RT. ItIt isis absolutelyabsolutely necessarynecessary toto useuse thisthis specificspecific antibodyantibody clone (MoBU-1), asas itit hashas nono crosscross reactivityreactivity withwith EdU.EdU. 2.2. Wash 33 × 150 µµLL wash bubuffer.ffer. × 3.3. Add 200 µµLL 1:200 Goat Anti-Mouse AlexaAlexa FluorFluor (555)(555) inin washwash bubufferffer toto eacheach samplesample andand incubateincubate 45 min at RT. 4. Wash 3 × 150 µL wash buffer. 4. Wash 3 150 µL wash buffer. 5. Perform× Click-iT reaction following the manufacturer’s instructions. 5. Perform Click-iT reaction following the manufacturer’s instructions. 3.5. RNase A Treatment and DAPI Staining. Time for Completion: 00:20 h 3.5. RNase A Treatment and DAPI Staining. Time for Completion: 00:20 h 1. Resuspend cells in 200 µL wash buffer containing 100 µg/mL RNase A and 1 µg/mL DAPI and 1. Resuspend cells in 200 µL wash buffer containing 100 µg/mL RNase A and 1 µg/mL DAPI and incubate for 15 min. incubate for 15 min. 2. Centrifuge and resuspend in 200 µL wash buffer. 2. Centrifuge and resuspend in 200 µL wash buffer. 3. CRITICAL STEP Transfer solution to a 5 mL round bottom polystyrene test tube with cell 3. strainerCRITICAL cap. It is STEP necessaryTransfer to filter solution the cell to asuspension 5 mL round through bottom the polystyrene tube cap to testavoid tube cell with clumps. cell strainer cap. It is necessary to filter the cell suspension through the tube cap to avoid cell clumps. 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

Methods 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 Table2; 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. Figure2 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

5 250K 5 10 10 Condition 1 200K 104 104 150K Condition 2 FCB FCB 3 3

10 DAPI (H) 100K 10 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 figure. Firstly,2 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 Table3. 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 × 6 Inefficient FCB detection FCB dilution (12 10 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 1. Use freshly prepared dilutions Insufficient FCB population 2. Cell type used requires 2. Optimize dilutions prior to use separation different dilutions

1. Increase washing time 1. Not enough washes after 3.2 and volume 2. Too many cells used Poor BrdU detection 2. Do not exceed 12 106 for labeling × cells total

1. Increase washing time 1. Not enough washes after 3.2 and volume 2. Too many cells used Poor EdU detection 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 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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