Use of Multicolor Flow Cytometry for Isolation of SpecificCell Populations Deriving from Differentiated HumanEmbryonic Stem Cells

Isabella Mengarelli, Andrew Fryga, and Tiziano Barberi

Abstract

Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specific typesof cells from a heterogeneous population of live cells. Here we describe a multicolor flow cytometrytechnique that uses five distinct cell surface antigens to isolate four live populations with different surfaceantigen profiles. These profiles were used to help distinguishing between neural and nonneural (the lens)ectoderm derivatives within a highly heterogenous population of differentiating human embryonic stemcells (hESC).

Keywords: Flow cytometry, hESC, Differentiation, Fluorochromes, Antibodies, Sorting-analysis

1 Introduction

Human embryonic stem cells lines were originally derived fromhuman blastocysts and shown to be pluripotent, therefore capableof generating tissues that derive from all three germ layers (1).Because of their origin, differentiated hESC cultures are heteroge-neous, nevertheless analysis of specific cell populations requires theisolation of the cells of interest. FCM provides the researcher with atool to analyze cell composition in differentiating hESC culturesand allows the isolation of target cells. In a FCMmachine a suspen-sion of cells passes through a laser beam that reads intrinsic char-acteristics like size, complexity, and autofluorescence. In additionto these parameters, this machine can analyze cells that express aninternal fluorescent reporter, have incorporated a fluorescent dye(as a probe) or have been stained with fluorochrome-conjugatedantibodies (2). Modern flow cytometers are high-speed machines,analyzing cells at >100,000 cells/s and sorting at >30,000 cells/s.The fluorescence measurement is scalable with the addition ofmultiple laser lines, up to 10 different wavelengths fromUltraviolet(UV) to Infrared (IR). The fluorescence is measured by up to 20detectors using bandpass filters spectrally matched to the targetfluorochromes. Thanks to the power of this instrumentation

Methods in Molecular BiologyDOI 10.1007/7651_2013_55© Springer Science+Business Media New York 2013

FCM-sorting can identify and isolate groups of cells that expressthe same, preselected profile of multiple fluorescent antigens (3, 4).The method we describe here relies on the ability of antibodies tospecifically recognize epitopes on the cells surface. Therefore, sinceno intracellular epitopes are examined, permeabilization of cells isnot required, allowing the collection of live cells. The primaryantibody (herein referred to as: pAb) can be either directly conju-gated to a fluorochrome or it can be recognized by a fluorochrome-conjugated secondary antibody (herein referred to as: sAb) thatmust recognize the pAb with high specificity. Primary antibodiesdirectly conjugated to fluorochromes are extremely useful tosimplify the procedure of cell immunolabelling in multicolorFCM-Sorting applications. When sAbs are used, they should beconjugated to a fluorochrome and specifically recognize the pAbsspecies or isotype (alternatively a biotin–streptavidin bond can beused to link a pAb to a fluorochrome). It is important to usefluorochromes with minimum spectral overlap into other detectors.

In this chapter we describe the use of multiple pAbs directedagainst five different surface molecules for the identification andisolation of four different live cell populations, with distinct surfaceantigen profiles. We show how the specific selection of the targetantigens allows the discrimination between neural and nonneural(lens) types of ectoderm derivatives, formed during early differen-tiation of hESC. We discuss the limitation inherent to the use of asAb together with multiple pAbs isotypes. Finally, we explain therationale followed in setting up the sorting gates for selection of thedesired population types by describing the gating strategies usedduring post-sort software (FlowJo) analysis.

2 Materials

2.1 Solutions

and Tools

for Preparation

and Immunostaining

of the Cells

1. DPBS solution (Dulbecco’s Phosphate Buffer Saline, withoutCalcium and Magnesium).

2. HBSS (Hanks’ Balanced Salt Solution modified, with phenolred, without calcium and magnesium, liquid).

3. Trypsin (0.25 % Trypsin-0.02 % EDTA Solution).

4. DMEM/F-12 (Dulbecco’s Modified Eagle Medium/ F-12).

5. FBS (Fetal Bovine Serum).

6. Sorting Buffer: for a tot volume of 100 ml mix 50 ml EDTA(0.02 %), 47 ml PBS, 2 ml FBS or FCS (Fetal Calf Serum), 1 mlPenicillin/Streptomycin solution (100�) and pass through a0.22 μm syringe filter. Work in sterile conditions in a biosafetycabinet. Store it at 4 �C.

7. Trypan Blue: 0.4 % in PBS.

8. Antibodies: Table 1.

Isabella Mengarelli et al.

Table1

Com

ponentsin

each

preparationtube

Tube

Label

Cells

Primaryantibody

Secondary

antibody

Target

antigen

Stock

concentration

Finaldilution

ofpA

baFinaldilution

ofsAba

Sam

ple(tube1

)Sam

ple

+Anti-p75,mouse

IgG1-PEb

Human

p75

CD271

100μg

1:100

Anti-c-m

et,mouse

IgG1-A

PCb

Human

c-met

(HGFR)

10μg

/ml

1:20

Anti-C

D44,rat

IgG2b-A

PC/Cy7

bHuman

CD44

200μg

/ml

1:100

Anti-H

NK-1,mouse

IgM

Goat

anti-m

ouse

IgM-A

F488

Human

CD57

(HNK-1)

200μg

/ml

1:150

Anti-C

D15,mouse

IgM-PBb

Human

CD15

Unkn

own

1:133

Control(tube2)

Unstained

+

Control(tube3)

PE

+Anti-p75,mouse

IgG1-PEb

Control(tube4)

APC

+anti-c-M

et,mouse

IgG1-A

PCb

Control(tube5)

APC-C

y7+

Anti-C

D44,rat

IgG2b-A

PC/Cy7

b

Control(tube6)

AF488

+Anti-H

NK-1,

mouse

IgM

Goat

anti-m

ouse

IgM-A

F488

2mg/ml

(forsA

b)

1:400

Control(tube7)

PB

+Anti-C

D15,mouse

IgM-PBb

ControlFMO

(tube8)

-APC

+AllprimaryAbexcept

anti-c-M

et-A

PC

Goat

anti-m

ouse

IgM-A

F488

ControlFMO

(tube9)

-AF488

+AllprimaryAbexcept

anti-H

NK-1

ControlFMO

(tube8and9)aresuggestedcontrolsifneeded

,PEphycoeritrin,APC

allophycocynin,AF488Alexa

Fluor488,PBpacificblue

a μlstock

antibody/

μlfinalvo

lumeofincubationbuffer

bDirectlyconjugated

antibody

Application of Multicolor Flow Cytometry-Sorting on hESC. . .

9. 18 Gauge (18 G), 40 mm (length) and 21 Gauge (21 G),40 mm (length) needles.

10. 20 ml syringe.

11. 40 μm cell strainer (for 50 ml tube diameter).

12. 15 and 50 ml tubes. 5 ml, round-bottom, polypropylene(12 � 75 mm) tubes.

13. Neubauer Cell Counting Chamber and Counter Tally.

14. Twister Shaker Platform (place it at 4 �C).

2.2 Solutions

and Tools

for Collection of Live

Cells

1. ITS medium: 985 ml of MilliQ distilled water, DMEM/F12powder (Gibco, one bag for 1 l medium), 1.55 g D-glucose,2.438 g NaHCO3, 50 mg human apo-transferrin, 5 mg humaninsulin (dissolved in 5 ml of 10 mM NaOH), 60 μl Na Selenite(0.5 mM stock in distilled water), 10 ml Penicillin/Streptomy-cin (100�). Assemble the component in a beaker with mag-netic stirring, transfer the solution under a biosafety cabinet,filter it through a 0.22 μm filter and cover the bottle withaluminum foil to protect from light. Store at 4 �C.

2. Y27632 Rock Inhibitor (10 μM final).

3. FGF2 (basic fibroblast growth factor).

4. EGF (epidermal growth factor).

5. Table centrifuge with swing bucket rotor and adapters for both5 ml round bottom and 15 ml tubes.

2.3 Equipment

and Post-sorting

Analysis Software

1. BD Influx (five lasers) flow cytometry sorter.

2. FlowJo software.

3 Methods

The following procedure can be applied to differentiating cellsgrowing in adherent conditions in one 6 cm cell culture dish. Thestarting culture can be scaled up if the populations of interest areknown to be exiguous. Work is done under a biosafety cabinet insterile conditions. Steps are carried out at room temperature (RT)unless otherwise specified.

3.1 Preparation

of Cells for

Immunostaining

in Suspension

1. Remove medium from the dish. Wash the adherent cells oncewith 3 ml PBS. Aspirate it. Add 2 ml HBSS and leave at roomtemperature (RT) for 20–30 min. Aspirate it (see Note 1).

2. Add 0.8–1ml Trypsin solution to the cells, transfer at 37 �C/5 %CO2 and incubate for 5 min. Under a biosafety cabinet use aP1000 pipetman (set at 1,000 μl) to gently aspirate and releasethe suspension of cells a few times onto the bottom surface ofthe dish until all cells are detached. Immediately add 3 ml of a

Isabella Mengarelli et al.

solution containing FBS to the cell suspension to inactivate thetrypsin. Transfer the suspension of cells into one 50 ml sterilepolypropylene tube. Complete collection of the cells from thedish by washing with 2–3 ml PBS and collect them into thesame 50 ml tube (see Note 2).

3. To ensure single cell suspension, pass the solution with cellsthrough a sterile syringe equipped with an 18 G needle. Repeatby passing the cells four times through the same syringe with a21 G needle. Place a 40 μm cell strainer over a new sterile 50 mlpolypropylene tube.Filter the entire volumeof the cell suspension.Finish collecting the cells from the old tube by using 5ml PBS andpass it through the strainer into the same new tube (see Note 3).

4. Centrifuge the cells collected in the 50 ml tube at 310 � g for10 min at RT.

5. Prepare new 15 ml polypropylene tubes in which you willdistribute the pelleted cells and label them as indicated inTable 1. The appropriate control tubes need to be prepared atthis time (see Note 4).

6. After centrifugation, discard the supernatant using a glass cap-illary pipette connected to vacuum suction. Tap the pellet toloosen it. Using a P1000 pipetman resuspend the pellet in800 μl of 4 �C-cold Sorting Buffer (see Note 5). Generate ahomogeneous-looking cell suspension by gently pipetting thecells up–down. Store the cell suspension on ice.

7. Count cells in the suspension by diluting an aliquot of thesecells (e.g. 10 μl) in Trypan Blue solution (e.g. 190 μl) and mixwell in a 1.5 ml tube. Load 10 μl of this cell suspension in ahemocytometer chamber. Cover the suspension with the cov-erslip being careful not to generate bubbles. Count the totalnumber of live cells (that do not internalize the Trypan Bluedye) in the four larger quadrants at the four angles of thechamber grid (¼n), under a 10� magnification objective. Cal-culate N ¼ total number of cells/ml (see Note 6).

8. Distribute 1–5 � 105 cells in each control tube (15 ml tubes,see Note 7) and adjust the final volume to 300 μl with 4 �C-coldSorting Buffer. Adjust the final volume of the sample tube to800 μl with Sorting Buffer (transfer it to a 15ml tube). The cellsof the control (tube 2, unstained) are ready for FCM analysis;transfer them to a 5ml, round-bottom, polypropylene tube andstore them on ice until the remaining cells will be ready.

9. Add the unconjugated pAbs at the concentration indicated inTable 1 (see Note 8) to the control (tube 6) and to sample(tube 1) and to the FMO control (tube 8, if this control isprepared). Incubate cells with the primary antibody for 30 minat 4 �C (cold room) positioning the tubes on a rocking platformset to produce a gentle movement of the cell suspension alongthe tube (see Note 8).

Application of Multicolor Flow Cytometry-Sorting on hESC. . .

10. Add 5 and 12 ml PBS in the control (tube 6) and sample(tube 1) respectively to wash away the unbound antibody.Centrifuge tubes at 310 � g for 5 min (see Note 9). Aftercentrifugation, carefully remove all the supernatant payingattention not to disturb the pellet. Resuspend the cell pelletsof the control (tube 6) and sample (tube1) in 400 μl and 800 μl4 �C-cold Sorting Buffer respectively. Use a P1000 pipetmanand gently resuspend the pellets to obtain a visually homoge-nous cell suspension.

11. Add the AF488-conjugated sAb to the cell suspension incontrol (tube 6) and sample (tube 1) at the concentrationindicated in Table 1. Minimize the exposure to light of thefluorochrome-conjugated antibodies (see Note 10). Incubatethe cells for 30 min at 4 �C on a rocking platform.

12. Wash away the unbound sAb by adding 5 and 12 ml PBS to thecontrol (tube 6) and sample (tube 1) respectively. Centrifugethe tubes at 310 � g for 5 min. Aspirate the supernatant fromthe two tubes as much as possible, paying attention not todisturb the pellet. Repeat the wash for the sample (tube 1).Resuspend the cell pellets of the control (tube 6) and sample(tube 1) in 300 and 800 μl 4 �C-cold Sorting Buffer respec-tively. The cells in control (tube 6) are ready for analysis,transfer them to a 5 ml, round-bottom tube, keep them onice, in the dark, until when the remaining cells will be ready.

13. Add the directly conjugated pAbs to all the remaining controltubes (3–5, 7 and 8, 9 if used) and to the sample (tube 1) at thedilution indicated in Table 1. Protect tubes form the light andincubate the cells for 30 min on the rocking platform at 4 �C.

14. Add 5 ml (to the control tubes) and 13 ml PBS (to the sample,tube 1) to wash away unbound antibodies and centrifuge themat 310 � g for 5min.Discard the supernatant and resuspend thecell pellets in 400 μl (control tubes) and 2ml (sample, tube 1) of4 �C-cold Sorting Buffer (see Note 11). Using a P1000 pipet-man set to maximum volume, pipet up–down the sample cellsuspension to break large, loosely formed, cell aggregates. Filterthis suspension through a 40 μmcell strainer positioned (kept inplace manually) directly on a 5 ml, round-bottom tube intowhich the cell suspension will be finally collected (see Note11). Transfer all cell suspensions of the control tubes into 5 mlround-bottom tubes. Put all tubes in ice and keep them pro-tected from light. Prepare the medium (ITS) necessary for col-lection of the desired subpopulation of cells already containinggrowth and survival factors (e.g. Rock Inhibitor 10 μM, FGF210 ng/ml, EGF 20 ng/ml). Put 1 ml of ITS medium in four5 ml, round-bottom tubes, for cell collection. Growth factorsfor specific subpopulations can be added to the tube in whichthat population will be collected. Keep all capped tubes on ice.

Isabella Mengarelli et al.

3.2 Sorting Settings

and Strategy

The control tubes are run in the following order. Tube2 (unstained), tube 7 (PB), tube 6 (AF488), tube 3 (PE), tube 4(APC), tube 5 (APC-Cy7), then followed by tube 1 (sample).Gating Strategy: dead cells and debris are excluded from furtheranalysis by plotting FSC (forward scatter) vs. SSC (side scatter)signals, cells in the gate (called “live cells”) in Fig. 1a are subse-quently analyzed in a FSC vs. FSC Int. plot. From this second plot,cells in doublets or triplets (with smaller FSC value) are excluded byselecting the cells gated as in Fig. 1b. Only these “single cells” arefurther analyzed in each tube. In our experiments we collect thefour populations indicated in Table 2.

1. A gate is defined around the single cells of tube 2 (unstained) indot plots for the following combinations of channels: APC vs. PE(combination a, Fig. 1c), APC-Cy7 vs. AF488 (combination b,Fig. 1d), PB vs. AF488 (combination c, Fig. 1e) (see Note 12).

Fig. 1 Analysis of the unstained cells. (a) definition of “live cells” gate (axes: FSC vs. SSC). (b) definition of“single cells” gate as subpopulation of “live cells” gate (axes: FSC vs. FSC Intensity). (c–e) definition of the“negative cells” gates in the following channels; (c): APC ¼ [640]660_20 vs. PE ¼ [561]605_40 (called“combination a.” (d): APC-Cy7 ¼ [640]750LP vs. AF488 ¼ [488]528_38 (called “combination b”). (e) PB[405] 460_50 vs. AF488 ¼ [488]528_38 (called “combination c”)

Application of Multicolor Flow Cytometry-Sorting on hESC. . .

2. Analyze the cells from the “single cells” gate of each single-color control tube (tubes 3–7) in a dot plot, the axis of whichincludes the fluorochrome of that particular single color tube(e.g. Fig. 2b). Draw the gate that defines the boundarybetween positive and negative cells for that fluorochrome(Fig. 2b; see Note 13).

3. Now analyze the sample cells (of tube 1) defining first the “livecells” and their “single cells” subpopulation (as shown above),then using a sequence of dot plots with the fluorochromecombinations used before (combinations a, b, c). In each plotdefine the gates only for the cells with the desired (indicated inTable 2) fluorochrome profile (Fig. 3, see Note 14). Aftercollection, cells can be further analyzed with different techni-ques. Upon further molecular analysis the cells of population 3resulted to be human eye lens cells (5).

Table 2Surface profile (by fluorochrome) of the sorted populations

Populations

Fluorochromes 1 2 3 4

APC-Cy7 + + � �APC � + + �PE � � � �AF488 � � � +

PB � � � +

Fig. 2 Analysis of cells in a single color control tube (example: APC-Cy7). (a)definition of “single cells” gate as subpopulation of “live cells” gate (not shown).(b) definition of negative cells (red gate) and positive cells (black gate) in thecombination of channels that include APC-Cy7 ¼ [640]750LP (“combination b”)

Isabella Mengarelli et al.

4 Notes

1. The PBS wash helps removal of dead cells and debris from thetop surface of the cells facilitating the action of the HBSSsolution, which, by depleting Ca2+ and Mg2+ ions, helps dis-rupting the interaction among cell adhesion molecules. Appli-cation of this solution is sometimes sufficient to release cells,but tissues in advanced stage of differentiation present more

Fig. 3 Analysis of cells in the sample tube. The flow of analysis goes from the central panel (“comb. a”¼combination a) following the black arrows to the side panels (“comb. b” and then “comb. c”). Cells in thecentral panel represent the single cells that are a subset of live cells (analyzed as in Fig. 1). Black arrowsindicate how cells from each black gate are further analyzed. Cells within the pink gates represent the desiredfinal subpopulations indicated in Table 2

Application of Multicolor Flow Cytometry-Sorting on hESC. . .

complex cell adhesion interactions that require enzymatic treat-ment to dissociate.

2. For cell sorting any tissue should be dissociated to a single-cellssuspension. If the action of enzymes is necessary, particular caremust be taken in choosing the enzyme because the extracellularportion of some cell membrane molecules may be completelydigested, therefore eliminating a potential target epitope of thepAbs (6). Different “gentler” enzymes are now commerciallyavailable (e.g. TrypLE Express, TypLE Select, Accutase), it isadvisable to experimentally test which enzymes are the mosteffective in disaggregating the cells with minimum damage.Incubation with Trypsin should be protracted for the shortesttime possible, it may be helpful to follow the detachment of thecells by eye or under 5� magnification at 2 min incubationintervals. Try not to protract trypsin digestion beyond 10 min.A fraction of cells is usually lost due to harsh treatment withtrypsin, which should be used only on though-to-dissociatetissue types. After mechanical dissociation through thepipette, mix well the FBS solution (3 ml DMEM/F12 contain-ing 10 % FBS) with the cell suspension to help inactivation ofthe enzyme.

3. Depending on the total volume of the cell suspension, choosesyringes with the appropriate capacity so that the entire volumeof cells can be aspirated at once. Needles with different dia-meters are commercially available, use a larger needle diameterfirst (to start disrupting the larger clumps) and then apply asmaller needle (to complete a finer cell cluster disruption). Atthis point most cells should be in single-cells condition. Inevi-tably some cluster remains, but they will be filtered out usingthe strainer.

4. In multicolor FCM experiments it is necessary to includemultiple control tubes, which include the following: (1)unstained control, (2) single color controls, and (3) Fluores-cence Minus One (FMO) controls. The unstained control isused to setup the flow cytometer. This control defines thebackground or autofluorescence of the cells thus setting theboundary for the “negative” cells (Fig. 1). Single color con-trols: each fluorochrome used will have a separately stainedcontrol. This will be used to compensate for spectral overlapinto other detectors and will aid in defining gating boundariesbetween “positive” and “negative” cells (an example inFig. 2). Although not performed in our experiment, if uponanalysis of fluorescence emission in any channel it resultsdifficult to precisely set the boundary between positive andnegative cells (because of a weak fluorescence signal) it canbe useful to add an FMO control tube, as indicated in Table 1.In this tube all fluorochromes are added with the exception of

Isabella Mengarelli et al.

the one that is in question. During analysis it will be clearer atwhat level to set the boundary for the “negative” cells in thatchannel (3). Possible FMO controls for this experimentare indicated in the last two rows in Table 1, they wouldhelp defining the c-Met+ and HNK-1+ cells during gatesdefinition in those channels.

5. The initial volume in which to resuspend the cell pellet can bechosen on the bases of knowledge, from previous experiments,of the expected number of cells that can be obtained from one6 cm dish of differentiating hESC. In our procedure20–30 � 106 cells were usually obtained from one dish. Aftercounting the cells we adjusted the volume of cell suspension tohave 2.5–3.75 � 106 cells in 100 μl Sorting Buffer for thestaining incubation of the “sample” tube.

6. It is advisable to start from a low dilution factor, e.g. 10 μl cellsuspension in 90 μl Trypan Blue, which corresponds to a dilu-tion factor of 10. If the number of cells in the quadrants is toohigh to clearly count the single cells, further dilute the suspen-sion. The number of cells/ml in your initial suspension can becalculated by the formula:N ¼ (n � 2,500 � dilution factor).Counting the cells helps evaluating how much of the initialsuspension to distribute in the control tubes and how manycells are present in the sample tube/s.

7. A minimum of 1 � 105 cells is usually sufficient in the controltubes to provide a clear signal for gates definition. However, tohelp formation of a well-compacted pellet (necessary to removemost of the antibody-containing supernatant after the follow-ing centrifugations) distribute about 2.5 � 105 cells in eachcontrol tube (up to a maximum of 5 � 105 cells).

8. The five target antigens were selected as follows. The CD271,CD15, and HNK-1 molecules are known to be expressed onneural progenitor and nervous system cells (7, 8), therefore theywere used for negative selection of the neural component withinpopulations 1–3 (Table 2). The c-Met and CD44 antigens areinstead expressed on human lens cells (a nonneural ectodermderivative) (9, 10) and used for positive selection of populationswith lens fate potential. Most pAbs used are directly conjugatedto a fluorochrome, except for the anti-HNK-1 antibody, forwhich the use of a fluorochrome-conjugated sAb was necessary.Since this sAbmay cross-react with the anti-CD15 pAb (becausedirected against the same pAb’s isotype) the staining protocolwas modified, as described below, to minimize cross-reaction.The final concentration of each pAb (Table 1) should be definedexperimentally. The AF488-conjugated sAb is directed againstany mouse IgM pAb therefore it may cross-react with theanti-CD15 pAb (also an IgM). To minimize the cross-reactionwe incubated the cells, in control and sample tubes, first with

Application of Multicolor Flow Cytometry-Sorting on hESC. . .

the anti-HNK-1 antibody alone, and then, after the wash,with the AF488-conjugated sAb. The unbound sAb was thenwashed away twice with PBS before incubation with theremaining pAbs. In absence of a rocking platform, incubationwith the pAbs can be performed by keeping the cells on ice(protected from light) for 30 min, providing manual agitationevery 5–10 min to avoid cell sinking. It is possible to spectrallydistinguish the emissions of propidium iodide (PI) and phyco-erythrin (PE) (11), therefore PI could be added to the samplecell suspension to help the selection of alive cells that excludethis dye. To avoid additional chemical stress to our cells ofinterest we avoided the use of PI.

9. If working with a large number of cells, a 50 ml tube may beused for the sample cells; in this case the centrifugation time canbe prolonged to 10 min. If cells still appear not well pelleted,centrifugation speed may be increased (e.g. to 350 � g).

10. To minimize the exposure to light turn off the light in thebiosafety cabinet and cover the ice buckets with the lid.

11. An ideal final cell suspension is 10 � 106 cells/ml, thus definingthe volume in which to resuspend the pelleted cells. However, atoo concentrated cell suspension may favor formation of smallcell aggregates that will be excluded by the sorting through thegating strategy, resulting in a decreased number of effectivelysorted cells. Additionally, cell aggregates may clog the sorternozzle incurring unwanted experimental delay. On the otherhand, the speed of sorting may be increased if the cell sample isnot toodiluted.The speedof sorting is also affected by the nozzlesize (“smaller” nozzle size generates more drops, acceleratingthe sorting speed compared to larger nozzle sizes).However, thenozzle size should be chosen in relation to the size of the cells.Ultimately, the optimal cell concentration will be determinedempirically. The small number of cells in the control tubes usuallydoes not form large aggregates; therefore, if the final cell suspen-sion is visually homogeneous, filtration of these cells can beavoided. For filtration of cell suspensions that are not toodense, 5 ml round bottom tubes, equipped with filter caps, arecommercially available. However, for dense or large volume ofcells we found it faster and equally effective to use 40 μm filterstrainers (for 50 ml tubes) on 5ml tubes. The cells of interest aredirectly collected in media supplemented with factors that sup-port their proliferation and survival (12–14).

12. Analyze the fluorescent signals by using dot plots. Anycombination of two channels at a time can be chosen foranalysis of the signals in each dot plot. Keeping in mind thefluorescent profile of the desired final populations (indicatedin Table 2) each dot plot will provide information about

Isabella Mengarelli et al.

which cells are negative (or positive) for each specificfluorochrome.

13. To distinguish a “positive” fluorescence signal derived fromcorrectly immuno-labelled cells from a “negative” signalderived from background fluorescence, we compared the signaldetected in each single color control tube to the signal given bythe unstained cells in that specific fluorochrome channel. Thiscomparison is made by transferring the gate defined on theunstained cells for each fluorochrome channel (as in point 1) tothe dot plot of each single color control.

14. The cells in each gate defined in dot plot “comb.a” will beseparately analyzed in dot plots “comb.b” and cells from eachgate in dot plots “comb.b”will be analyzed in dot plots “comb.c.” To define the boundaries between positive and negativecells for each fluorochrome use the gates defined for theunstained and single color control cells. In using these previ-ously defined gates, the investigator can apply at his/her owndiscretion different levels of “stringency,” that is how close anew gate will be to the gate that defines the “negative” cells.

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4. Andrew L. Laslett, Andrew Fryga, Martin F.Pera (2007) Flow cytometric analysis ofHuman embryonic stem cells. Human stemcell manual, 1st edn., Elsevier Inc., pp 96–107

5. Mengarelli I, Barberi T (2013) Derivationof multiple cranial tissues and isolation oflens epithelium-like cells from human embry-onic stem cells. Stem Cells Transl Med2:94–106

6. Pokutta S, Herrenknecht K, Kemler R et al(1994) Conformational changes of the recom-binant extracellular domain of E-cadherinupon calcium binding. Eur J Biochem223:1019–1026

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10. Saika S, Kawashima Y,Miyamoto Tet al (1998)Immunolocalization of hyaluronan and CD44in quiescent and proliferating human lensepithelial cells. J Cataract Refract Surg24:1266–1270

11. Corver WE, Cornelisse CJ, Fleuren GJ (1994)Simultaneous measurement of two cellular anti-gens and DNA using fluorescein-isothiocyanate,R-phycoerythrin, and propidium iodide on astandard FACScan. Cytometry 15:117–128

12. Kanemura Y, Mori H, Nakagawa A et al (2005)In vitro screening of exogenous factorsfor human neural stem/progenitor cell prolif-eration using measurement of total ATP con-tent in viable cells. Cell Transplant 14:673–682

13. Lovicu FJ, McAvoy JW (2005) Growth factorregulation of lens development. Dev Biol280:1–14

14. Watanabe K, UenoM, Kamiya D et al (2007) AROCK inhibitor permits survival of dissociatedhuman embryonic stem cells. Nat Biotechnol25:681–686

Application of Multicolor Flow Cytometry-Sorting on hESC. . .