Gating strategies on:-Cell population

(expressed in Log or Lin)

-Positive/negative cells (gaps between 2 regions)

1 2 3 4 5 6 7

20

40

60

80

100

EC50 => Mobicyte

Participant

EC-5

0 [

Cd

] (µ

M)

Δ = 24,66 µM

1 2 3 4 5 6 7

40

60

80

100

120

140

160

Unkown [Cd] => MOBICYTE

Participant

[Cd

] (µ

M)

Δ = 31,93µM

Expected values 70µM

1 2 3 4 5 6 7

20

40

60

80

100

EC50 => Participant

EC-5

0 [

Cd

] (µ

M)

Participant

Δ = 39,49 µM

1 2 3 4 5 6 7

40

60

80

100

120

140

160

Unkonw [Cd] => ParticipantParticipant

[Cd

] (µ

M)

Expected values 70µM

Δ = 100,57 µM

In flow cytometry, does results harmonization require the same equipment? Case of cell viability.

Flow cytometry allows analysis of cellularparameters or functions in order to obtain single-cell and populational data with a statisticaldimension. University of Reims and INERIS, haveset up a mobile environmental flow cytometryfacility: MOBICYTE. This platform is dedicated toapplications in ecotoxicology, in functionalecology and in integrative ecophysiology.

Many cellular functions can be studied byflow cytometry by using a wide variety ofcommercialised fluorescent probes. As manyresearch teams work on identical cellularresponses (i.e. cytotoxicity tests), variations ofprotocols and methodologies for cytometricanalyses can lead to misinterpretations withconsequences for toxicological risk assessment.

In this context, we have estimated the abilityof different groups to assess cadmium EC-50with a same sample set by the use of flowcytometry technique. This inter-lab calibrationimplicated different flow cytometry equipment.

BD Accuri C6

Beckman Cyan

Millipore Guava

8HT

Beckman Gallios

Beckman Cell Lab Quanta

Millipore Guava

BD Accuri C6 Acquisition

Samples were analysed on 7 different cytometerswith the software dedicated to equipment, we asked to:

- Gate in cell population (FSC/SSC)- Acquire 20 000 cells in this region- Split positive and negative cells in FL-1 (+/-)

- Determine EC50 of cadmium (their own method)

- Determine unknown concentration (their own method)

II

Participant 1 2 4 5 6 7

Reasoning on live cells 51,54 111,34 85,21 97,43 161,08 203,84

Reasoning on dead cells 50,79 65,63 61,96 71,94 78,18 90,27

Rioult D.a-c, Bados Nilles A.a-c, Serpentini A.d, Gagnaire B.e, Auffret M.f, Brousseau P.g, Le Foll F.h, Betoulle S.c

a Université de Reims Champagne-Ardenne / INERIS, Plateau MOBICYTE, UFR Sciences Exactes et Naturelles, BP 1039, 51687 Reims cedex 2.

b INERIS, UMR-I 02 SEBIO, Parc Technologique Alata, B.P. 2, 60550 Verneuil-en-Halatte, France.

c Université de Reims Champagne-Ardenne, UMR-I 02 SEBIO, UFR Sciences Exactes et Naturelles, BP 1039, 51687 Reims cedex 2.

dNormandie Université, Caen; UMR BOREA, MNHN, UPMC, UCBN, CNRS-7208, IRD-207, SFR ICORE, IBFA, Caen, France.

eInstitut de Radioprotection et de Sûreté Nucléaire (IRSN), laboratoire d’écotoxicologie des radionucléides, Saint-Paul lez Durance, France.

fLEMAR, UMR 6539, Institut Universitaire Européen de la Mer, 29280 Plouzané, France

gInstitut National de la Recherche Scientifique-Institut Armand-Frappier, Laval, Québec, Canada,

h Université du Havre, UMR-I 02 SEBIO UFR Sciences et Techniques, 76600 Le Havre, France

SEBIO-ULH, Le Havre

INERIS-URCA MOBICYTE

I THP-1

Samplepreparation

Treatment Staining

URCA-INERIS MOBICYTE

UMR LEMAR, Brest

IRSN, Saint-Paul lez Durance

UMR BOREA, Caen

0µM, 10µM, 50µM,

100µM, 500µM, 1000µM

???ctrl

Human monocyticcell line

Sending by postal way

…

Live/Dead® Fixable Green StainCadmium 16h.

A single cell preparation was perfomed. Cells coming from a single experiment were sent to participant.

0,1 1 10 100 1000 10000

0

20

40

60

80

100

RAW data analyzed by each participant with their own equipment.Gating and data extraction were performed on flow cytometer dedicatedsoftware. In many cases, EC-50 values were determined with Excel™ macroRegTox.

Summarized EC-50 curves created with all data analysis through MOBICYTE facilities

(Probe flurorescence : λex=495 nm / λem=520 nm)

Analysis

III

RAW data analyzed by MOBICYTE.After acquisition in each lab, RAW data (FCS files) were sent to Mobicyte. Gatingand analysis were performed with FlowJo© (FlowJo, LLC). EC50 and unknownconcentration were determined with SigmaPlot© (Systat Software Inc.)

Some variabilities identified After discussion with partners, it turns out that the main source of variability was in post-acquision analysis. This step is the one

involving the most intervention of man.

Values of EC-50 (µM) determined with RegTox. Application on live cells or dead cells give different values of EC-50.

Excel™ Macro RegTox

[Cd] (µM)

% o

f d

ead

cells

Conclusion: Despite the diversity of flow cytometers, results were rather homogeneous.

Variability lies mainly in the post- acquisition analysis (human intervention)To analyze cytotoxicity data, some conclusions and recommendations can be listed:

• No need of specific equipment• Acquire enough cells (20 000 events on cells gate).• Be binary and empirical in viability analysis.• If probe detects dead cells, work only with dead

cells percentage.