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The Flow Cytometryand Cell Sorting platform
of the Cancer Research Centre of Lyonhttp://192.168.143.114/78-Cytometrie.crcl.aspx?
language=fr-FR- - -
Centre Léon Bérard
General rules and procedures
The Flow Cytometry and Cell Sorting platform of the Cancer Research Centre of Lyon (CRCL) – Léon Bérard Centre (CLB) is available to the CRCL research teams, to teams belonging to the “Recherche de Transfert et d’Innovations”, and to public or private users (academic or industrial) outside of the CRCL/CLB.The platform includes both analysers and cell sorters.
The platform is managed by a researcher and a technical supervisor.
The Planning Committee (“Comité de Pilotage”) oversees the evolution of the platform. It comprises members of the Board of Directors for Research (Direction de la Recherche), platform supervisors, as well as acting members of the main teams using the platform (see appendix 1).
1. Supervisors1.1.Scientific supervisor
Christophe CAUXLYON CANCER RESEARCH CENTRECentre Léon BérardBâtiment Cheney D (3rd floor)28 rue Laennec69373 LYON Cedex 08 Tel : 04 78 78 51 39 (direct line)Fax : 04 78 78 27 20
1.1.1. Missions To oversee the technical supervisor. To organise and host the Planning Committee. To ensure proper coordination with the SFR (Lyon-Est based Research Health
Federation). In collaboration with the Planning Committee and the SFR, to implement the
technological and organisational evolutions of the platform. To be responsible for finding the resources necessary to purchase new equipment.
1.2.Technical supervisorIsabelle DURANDLYON CANCER RESEARCH CENTRECentre Léon BérardBâtiment Cheney D (Ground floor)28 rue Laennec69373 LYON Cedex 08 Tel : 04 78 78 29 12 (direct line)Fax : 04 78 78 27 20
1.2.1. General missions To ensure the regular maintenance of the machines. To advise users (on the choice of fluorophores, on controls). To give access to standard protocols via the server (see appendix 2). To watch out for technological advances. To watch out for recent advances in data-processing. To regularly monitor and update relevant information (new products or techniques). To organise preventive maintenance of the equipment with the supplier. To take part in the selection of new equipment, whenever a turn-over becomes
necessary. To update the general rules and procedures. To record and supply the data necessary for the billing of the services provided. To collaborate with other members of the SFR to setup and organise a Technical
Platform cluster dedicated to Cytometry in the Lyon-Est area. To make sure the general rules and procedures are applied by all users. In the
event of non-compliance to these rules and regulations, users will no longer be permitted to gain access to the platform. The platform supervisors will also relay the information to the user’s supervisor.
Update the platform’s intra and internet pages.
1.2.2. Analysers To plan training sessions. To train users in order to optimise their use of the analysers. To advise users, if necessary, on the calibration of the analysers (users must
however analyse their own samples). To help users, if necessary, to interpret their data. To ensure compliance with the general rules and procedures and proper usage of
the equipment.
1.2.3. Cell sorters To program cell sorting parameters. To make sure the cells have been sorted.
2. Missions of the Planning Committee To define the operating conditions of the platform. To enforce / modify the general rules and procedures. To discuss the financial report. To define the billing rates. To validate the investment projects.
3. Localisation
The following equipment is located on the ground floor of the Cheney D building:- the FACSCalibur 4 colours (room 008)- the CyAn ADP (room 008)- the LSR Fortessa (room 008)- the LSR Fortessa HTS (room 002)- the cell sorter (room 009 – entrance through room 008)- the office of the technical supervisor (room 007)- the data analysing computers (room 002)
4. To gain access to the analysers and to the computer roomYou need a CLB/CRCL badge to gain access to rooms 002 and 008.
5. Operating conditions for the cytometers5.1.Analysers
Anyone wishing to use an analyser must be trained by the technical supervisor, beforehand, even if he/she used a similar cytometer in another laboratory.
So as to limit the training of occasional users, short-stay trainees (< 3 months) will no longer be trained and will thus no longer be authorised to use these machines.
Users should participate in the training sessions specific to a given equipment, since each machine is different, according to the equipment they will be using.
The training sessions are not free (for the price list, see appendix 4). Anyone wishing to be trained should organise a session with the technical supervisor. The users have to have read and completed the form (see appendix 3) stating that
they accept the terms and conditions of the general rules and procedures, as well as dated and signed the form.
5.2.Cell sorters
The cell sorters can only be used by the technical supervisor.
6. Procedures6.1.Health and Safety Rules
6.1.1. What is compulsory Users should wear a lab coat and gloves at all times. The waste should be sorted and dealt with according to the instructions outlined by the
technical supervisor during the training sessions. The utensils/glassware/samples you may have brought into the cytometry rooms
should be removed at the end of your session. Any hazardous spillage should be cleaned up using a disinfectant. Any operating mistakes/problems or equipment malfunction should be reported to the
technical supervisor.
Any consumables which may run out should also be reported to the technical supervisor.
6.1.2. What is forbidden To smoke, drink or eat on the platform. To use the telephone whilst wearing gloves.
6.1.3. What is strongly unadvised The use of mobile phones in the vicinity of the machines, due to the possibly
contamination of the workspace surrounding the cytometer (possible contamination of cells or chemical products, intercalants).
6.2.Operating procedures for the analysers It is imperative that users should follow the proper procedures for switching on,
cleaning and switching off the machines. These procedures were given to you during your training.
If the users have not been fully trained, the technical supervisor will conduct the initial steps and calibrations, and the users will then run their own samples in complete autonomy, knowing that they can always ask for help and advice if need be.
It is absolutely forbidden to “tinker” or “tamper” with the analysers.
7. Booking system for the analysers and schedule for the cell sorters7.1.Analysers
7.1.1. Booking schedule for sample analyses The platform uses the online SFR booking system. In order to use a cytometer, a user has to have created a user account. Once the
user has been trained his/her user account will be validated and he/she will be authorized to use the machines.
The document entitled “Presentation of the flow cytometry booking system” (accessible via the V:\CYTOMETRIE EN FLUX\), contains all of the information necessary to create a user account, to be authorised to use a cytometer, to book/cancel a time slot on the schedule of a particular machine.
The user should book slots on the analysers as early as possible. A booked time-slot should not exceed 3 hours. If the user requires the machine for
more than 3 hours, he/she should leave a 30-minute (or even 1 hour) slot free at the end of the 3 hours and before his/her next slot, to enable other users to conduct short analyses.
If a user wishes to cancel a time-slot, he/she should: cancel his/her booking (see the presentation of the flow cytometry booking
system) warn the next user booked on the schedule
make sure the equipment will be cleaned properly if he/she was the last person booked on that day and possibly clean the machine if no one else is using it.
If a cytometer is not cleaned and switched off properly, a message will be sent to the user and to his/her team leader and a fine equivalent to 10 hours booked on the machine will be billed to the team of: the last user if he/she actually ran some samples the last but one user, if the last user cancelled his/her time slot before the end
of the time slot of the last but one user the last user if he/she cancelled his/her time slot after the time slot of the last
but one user ended in the latter two cases, the data saved on the online booking schedule will be
used as evidence. If a user has not cancelled his/her booking, the time booked will be billed anyway. A user who starts late must leave the equipment on time for the next user who
wishes to start on time.
7.1.2. Analyser operating time The computer booking system and the recording of the time spent on the machines
(machine “operating time”) are linked. The users should include in their “operating time”, time spent setting up/calibrating
the equipment, i.e. choosing the optimal acquisition parameters (controls, compensation), as well as the time spent cleaning the equipment.
Quality control procedures (CS&T for the Fortessa and Calibrite for the FACSCalibur 4C) are not included in the “operating time”.
Time spent analysing results is not included in the “operating time”. The machines should principally be used for data acquisition. Data analysis should
be conducted on specific “data analysis” computers.
7.2.Cells sorters7.2.1. Booking schedule for cells sorters
Several limitations should be taken into account when planning to conduct cell-sorting experiments: The schedule for the analysis of clinically extracted breast cancer samples is
available a week in advance (generally on the Thursdays) The reception of some leukemic samples, following surgical operations, is,
however, unpredictable and their analyses are urgent Users should book their time slots well in advance by contacting the technical
supervisor. If you need to cancel a time slot, you should also contact the technical supervisor
as soon as possible so that the time slot may be given to another user.
The user should send an e-mail with a completed “before using the cell sorter” data sheet to the technical supervisor one day prior to his/her time slot, at the latest.
The user should systematically send a “cell sorting results" data sheet to the technical supervisor, at the end of the primary cell culture or within 15 days for cell lines. These data are invaluable for confirming that the cell sorter has not been contaminated/that it is still sterile.
7.2.2. Cell sorter operating time The operating time of the cell sorters is recorded by the technical supervisor. This time includes the time spent calibrating the machine, i.e., choosing the optimal
acquisition parameters (controls, compensation) and the time spent analysing the cells.
The time spent analysing the results is not included.
8. Data storageUsers are requested to store a minimum of data on the hard drive of the computers linked to the cytometers. Indeed, in the event of a breakdown, the data could be definitely lost.In the case of the Fortessa, the data base should not exceed 10 Gb. Indeed, beyond this threshold, new data will be written over old data (the data of other users will be definitely lost).The group leaders/team leaders should provide their members with external storage units (ZIP, CD, DVD, external hard drives) or space on a storage network (N or V for example in the CRCL).The storage of data on the NAS:\CYTOMETRIE (N internal network) is only permitted for members working at the CRCL: previous CRCL members having left the laboratory should store their data on a different storage medium.
8.1.The Mac of the four-color capacity FACSCaliburThis computer has:
- A USB portAnd it is connected to the internal CRCL N (NAS) network.
8.2.The PC linked to the CyAn ADPThis computer has:
- A USB port- A CD/DVD reader/writer
8.3.The PC linked to the LSR FortessaThis computer does not have an antivirus (since they are all incompatible with the software). It is therefore absolutely forbidden to insert USB drives or external hard drives into the ports of this computer. It is also forbidden to access internet from this computer. Anyone breaching these rules will not be authorized to use the Fortessa for a period of 15 days. The data should systematically be exported to the data analysis computer, which is connected to the internal N (NAS) network.
8.4.The PC linked to the LSR Fortessa HTSThis computer does not have an antivirus (since they are all incompatible with the software). It is therefore absolutely forbidden to insert USB drives or external hard drives into the ports of this computer. It is also forbidden to access internet from this computer. Anyone breaching these rules will not be authorized to use the Fortessa for a period of 15 days. The data should systematically be exported to the data analysis computer, which is connected to the internal N (NAS) network.
9. Data analysis computers3 computers are set up for analysing you data:
- 1 Mac set up to analyse data obtained on the four-color capacity FACSCalibur, using the: o CellQuest Pro softwareo Or ModFit software (to analyse cell cycle and cell proliferation – CFSE)
- 1 PC has the:o DiVa software (to analyse data obtained on the LSR Fortessa)
- 1 PC has the:o DiVa software (to analyse data obtained on the LSR Fortessa)o And the FlowJo software (to analyse data obtained on all cytometers)
As is the case for the computers linked to the cytometers, you are requested not to store large data sets on these computers. All data should be transferred to the internal NAS network.
10. Billing of the cytometer usage
In order to cover the cost of maintaining the equipment, users are billed for the time they spend on the machines.The rates, listed in appendix 4, are revised each year.Bills are sent every trimester to the different teams using the equipment.
1. AnalysersThe “operating time” used to calculate the bills is based on the time slots booked on the electronic schedule.
2. Cell sortersThe reckoning is as follows:
- A cell sorting time slot under 1h is actually billed as an 1h slot, and includes: The time spent switching on the machine, the calibration and the time spent
switching off the machine The consumables used (fluorescent beads for the calibration, de-contamination
reagents)
- A cell sorting time over 1h will be billed according to the actual time spent on the machine
11. Information dissemination
Only the users having created a user account will receive information concerning the cytometers (new equipment/software/reagents, breakdowns, absence of the technical supervisor…) (see also: Booking system for the analysers and schedule for the cell sorters\Analysers\Booking schedule for sample analyses)
12. Electronic file “Flow Cytometry”
An electronic file “Flow Cytometry” is available in French for users under V:\CYTOMETRIE EN FLUX (internal CLB network).Appendix 2 includes a short description of the information available in this file.
Appendix 1
Members of the planning committee
Members of the Research directing committee
Marina ROQUES-RAIMONDIAdministrative Director – CRCL
Julie POURCHET-PIETRYKAAdministrative Deputy Director – CRCL
Technical supervisor of the Flow Cytometry and Cell Sorting platform
Isabelle DURANDCRCL
Acting members of the teams mainly using the platform
Christophe CAUXCRCL – Targeting of the tumor and its immune environmentCharles DUMONTETCRCL – Anticancer Antibodies
Véronique MAGUER-SATTACRCL – BMP, tumoral niche and resistance
Julien MARIECRCL – TGF and immune evasion
Anne-Pierre MORELCRCL – EMT and cancer cell plasticity
Toufic RENNOCRCL – Innate Immune Signaling and Oncogenesis
Birke BARTOSCHCRCL - Pathogenesis of Hepatitis B and C infections
Virginie PETRILLICRCL – Inflammasome and cancer
Jean-Guy DELCROSCRCL – Dependence receptors, cancer and development
Acting members of the CRCL plateforms
Serge MANIE (platform manager and scientific supervisor)CRCL – Innate Immune Signaling and Oncogenesis
Christophe VANBELLE (head of the imaging platform)CRCL – Imaging platform
Appendix 2
Electronic file “Flow Cytometry”This file is available in French for users under V (internal CLB network).
1. Information on the Antibodies
2. Information on Fluorophores
3. FACSAria III (sorter)3.1.1. Before using the cell sorter
A data sheet detailing the experiments you will be conducting on the cell sorter (excluding the sorting step) should be completed and sent to the technical supervisor, the day before your experiment at the latest. An extension is given to users requesting the urgent analysis of leukemic samples.
3.1.2. Before sorting the cells A data sheet specific to the sorting of cells should be completed and sent to the technical supervisor, the day before your experiment at the latest. An extension is given to users requesting the urgent analysis of leukemic samples.
3.1.3. Advice for sorting cells Technical advice for sorting cells.
3.1.4. Cell sorting results A data sheet specific to the cell sorting results (cell culture) should be sent to the technical supervisor by e-mail at the end of the cell culture (for primary cells) or within 15 days (for cell lines) of the cell sorting experiment. This information enables us to detect contaminations as soon as they occur and to make sure that the sorter is sterile for other users.
4. Technical data sheets
5. Fluorophores and cytometersCompatibility between the different fluorophores and the cytometers used on this platform. 6. Standard protocols
7. Cleaning instructions for the Fortessa
8. Presentation of the booking system
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9. General rules and procedures
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Appendix 3
Statement that you agree with the general rules procedures
Please make sure you return this document to the technical supervisor, electronically completed (to make it more legible) and signed.
I (first name (s) and SURNAME) (1) : Position held : Member of the team : Led by (2) :
Declare having read the above mentioned general rules and procedures of the Flow Cytometry and Cell Sorting platform of the Cancer Research Centre of Lyon –Léon Bérard Centre, and make the commitment to follow these guidelines. Declaration made in : On the (date) :
Signature :
(1) : Your first name(s) and SURNAME(2) : First name (s) and SURNAME of your scientific supervisor (team leader)
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Appendix 4
Billing rates
Rates for 2015
1- Cytometer-“operating time”SFR Lyon-Est (CRCL – CLB –
SFR)SFR Gerland
External academic users SFR Lyon-Est
and SFR Gerland
Industrials
Analysers hour € HT(TTC)
30(36)
75(90)
126(151)
Sorters hour € HT(TTC)
75(90)
126(151)
250(300)
2- Training
The training of users will be billed 120 € HT / per user / per machine.
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