1 2 3 4 5 6 7 EC50 => Mobicyte...URCA-INERIS MOBICYTE UMR LEMAR, Brest IRSN, Saint-Paul lez Durance...
1
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 EC50 => Mobicyte Participant EC-50 [Cd] (μM) Δ = 24,66 μM 1 2 3 4 5 6 7 40 60 80 100 120 140 160 Unkown [Cd] => MOBICYTE Participant [Cd] (μM) Δ = 31,93μM Expected values 70μM 1 2 3 4 5 6 7 20 40 60 80 EC50 => Participant EC-50 [Cd] (μM) Participant Δ = 39,49 μM 1 2 3 4 5 6 7 40 60 80 100 120 140 160 Unkonw [Cd] => Participant Participant [Cd] (μM) 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 cellular parameters or functions in order to obtain single- cell and populational data with a statistical dimension. University of Reims and INERIS, have set up a mobile environmental flow cytometry facility: MOBICYTE. This platform is dedicated to applications in ecotoxicology, in functional ecology and in integrative ecophysiology. Many cellular functions can be studied by flow cytometry by using a wide variety of commercialised fluorescent probes. As many research teams work on identical cellular responses (i.e. cytotoxicity tests), variations of protocols and methodologies for cytometric analyses can lead to misinterpretations with consequences for toxicological risk assessment. In this context, we have estimated the ability of different groups to assess cadmium EC-50 with a same sample set by the use of flow cytometry technique. This inter-lab calibration implicated 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 cytometers with 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. d Normandie Université, Caen; UMR BOREA, MNHN, UPMC, UCBN, CNRS-7208, IRD-207, SFR ICORE, IBFA, Caen, France. e Institut de Radioprotection et de Sûreté Nucléaire (IRSN), laboratoire d’écotoxicologie des radionucléides, Saint-Paul lez Durance, France. f LEMAR, UMR 6539, Institut Universitaire Européen de la Mer, 29280 Plouzané, France g Institut 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 Sample preparation Treatment Staining URCA-INERIS MOBICYTE UMR LEMAR, Brest IRSN, Saint-Paul lez Durance UMR BOREA, Caen 0μM, 10μM, 50μM, 100μM, 500μM, 1000μM ??? ctrl Human monocytic cell line Sending by postal way … Live/Dead® Fixable Green Stain Cadmium 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 dedicated software. In many cases, EC-50 values were determined with Excel™ macro RegTox. Summarized EC-50 curves created with all data analysis through MOBICYTE facilities (Probe flurorescence : λ ex =495 nm / λ em =520 nm) Analysis III RAW data analyzed by MOBICYTE. After acquisition in each lab, RAW data (FCS files) were sent to Mobicyte. Gating and analysis were performed with FlowJo© (FlowJo, LLC). EC50 and unknown concentration 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) % of dead 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.
Transcript of 1 2 3 4 5 6 7 EC50 => Mobicyte...URCA-INERIS MOBICYTE UMR LEMAR, Brest IRSN, Saint-Paul lez Durance...
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.
