Use of the cytometry platform - Rules of Procedure. Use of the cytometry platform University of Bordeaux - CNRS UMS INSERM US 005

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1 Use of the cytometry platform University of Bordeaux - CNRS UMS INSERM US 005 This version is: Written/Revised by: Vincent Pitard, IR, Technical Manager Verified by: Approved by: Anaëlle Stum, Assistant Engineer Julie Déchanet, Director UMR5164 Validated by: Julie Déchanet, Director UMS 3427 Table of Contents 1 Services offered...2 *Assisted session:...2 *Provision of equipment after training:...2 *Feasibility study:...3 *Scientific collaboration: Valuation/publication...3 *Materials and Methods...3 *Acknowledgements Instruments available Reservation and time of use Sample acquisition and machine utilization Data storage and analysis of results Mandatory health and safety rules to be applied Invoicing PPMS account creation request and agreement...6 1/12/2017 version n 15 1

2 1 Services offered Use of the cytometry platform - Rules of Procedure Within the platform, four services are offered to you: Assisted session, Provision of equipment after training, Feasibility study, Scientific collaboration. For any request, please send an to cytometrie@immuconcept.org or call and ask Vincent Pitard or Anaëlle Stum. It is most often followed by a first visit to determine the conditions of the service and specify the conditions of use of the platform. The access codes of the premises will be given to you. A request for a PPMS account must be made online to book instruments by filling the questionnaire at the end of this document. Access to the platform is available 24 hours a day for autonomous users. The technical staff is present from 8 am to 6pm and does not make appointments outside these hours. Further information can be found on the TBMcore web site *Assisted session: The assisted session concerns the work carried out by the platform on behalf of a user requiring specific use of analysers (less than two hours in one month or four times a year) or for all nonautonomous cell sorting. It implements techniques or know-how mastered by the platform (known protocols, standard experimental conditions, not requiring any special adaptations or developments). The work done in this type of service is not inventive. This type of service is only provided by the platform staff. The cytometer reservation is made by the user assisted by platform staff. For cellular sorting, reservations are made directly by the users but subject to the agreement of the platform staff. They must be booked no later than Friday of the week before sorting and no earlier than one month in advance. Last minute reservations are not accepted. *Provision of equipment after training: The provision of the equipment requires the prior training of users by platform staff. The provision of equipment after training is proposed when the user's needs necessitate repeated use of equipment and when the user wishes to invest in learning the technique. This training results in the acquisition of autonomy allowing the user to book the equipment on which he has been trained. The training is generally divided into 3 sessions of one hour each. During the first one, the person in charge explains the operation of the device and the software by passing the tubes of the future user who notes and tries to remember the progress of the operation. For the second session, the user prepares the machine and his experience under the supervision of the person in charge who corrects it and lets him pass his tubes. Finally, at the third session, the person in charge assesses the autonomy of the future user and gives him/her the last advice and authorization to pass his/her samples alone. The validity of the autonomy is reviewed at least once a year according to the following criteria: frequency of use and respect for the equipment, compliance with health and safety rules and instructions for use of the platform. Failure to observe the rules of use may result in loss of autonomy for the user. The reservation of the equipment is carried out by the autonomous user under his/her name. 1/12/2017 version n 15 2

3 *Feasibility study: A feasibility study is carried out when a request does not correspond to a service or equipment provision and requires a more detailed analysis of the user's needs (testing...). Its duration is rather short (less than 1 month). It can be used to test several equipment and/or protocols and may precede more in-depth scientific collaboration, service provision or training for provision. The future user must be present during the tests. The reservation is made by the platform staff on behalf of the user or requesting laboratory. *Scientific collaboration: Scientific collaboration is designed to carry out studies and experiments on a specific theme, in direct collaboration with a user. Collaboration is implemented when the user's needs may require from the platform to adapt existing protocols or to implement specific developments. The commitment to scientific collaboration requires the prior agreement of the platform's technical and scientific managers. 2 Valuation/publication In order to promote the platform, users mention the University of Bordeaux's cytometry platform in their publications and thank the technical staff for their assistance. The feasibility study and scientific collaboration may lead to co-signature of the publications. *Materials and Methods FACS data were acquired using a LSR Fortessa 2-Blue 6-Violet 3-Red 5-YelGr laser configuration (BD Biosciences) a CantoII 4-Blue 2-Violet 2-Red laser configuration (BD Biosciences) a Canto 4-Blue 2-Red laser configuration (BD Biosciences) Cell were sorted using a FACSAria 2-Blue 6-Violet 3-Red 5-YelGr, 2 UV laser configuration (BD Biosciences) in flow cabinet FACSAria 5-Blue 2-Violet 2-Red laser configuration (BD Biosciences) Flow cytometry analysis were performed using Diva 8 (BD Biosciences) and FlowJo (Tristar) softwares *Acknowledgements We thank Anaëlle Stum and Vincent Pitard for technical assistance at the Flow cytometry facility, CNRS UMS 3427, INSERM US 005, Univ. Bordeaux, F Bordeaux, France 3 Instruments available See regular updates on the cytometry page of the TMBCore platforms website 1/12/2017 version n 15 3

4 4 Reservation and time of use Use of the cytometry platform - Rules of Procedure Reservations are made at the earliest (maximum 30 days in advance). Minimum reservation period is one hour for each of the instruments. Reservations are made through PPMS web application with your login and password. The instrument reservation is for sample acquisition time only. Each instrument is equipped with a tracker which records in real time the use of Diva software and allows to adjust the invoicing (overruns) once you have logged in. Do not forget to log off once finished. CantoII and Fortessa reservations of more than two hours between 9am and 6pm on the same day is not possible unless you notify platform staff. Outside the opening hours of the service (9H-18H), the time of use is not limited but requires the agreement of the platform staff and to warn the PC security at extension or after 20H All reservations must be cancelled as soon as possible by the user as soon as they are confirmed (problem of cells, samples, etc.) and warn users before or after the slot is released. Any unbooked instrument or any unused reserved analyser that has not been cancelled at least one hour prior use for analysers and at least 12H prior use for sorters will be charged and a 100% penalty will be applied. When the cancellation corresponds to the last slot of the day, the user must make sure that the machine will be stopped and must come to do it himself. Any machine left on at night may result in permanent exclusion from the platform of the offending user. 5 Sample acquisition and machine utilization For any experiment, the laboratory notebook of the machine is filled with the user name, the time of start and end of acquisition, the cellular types analysed, the number of samples and the type of labelling. All procedures for cleaning, start up or shutting down the equipment are also recorded. All incidents that occur must be reported by clicking Report Incident in the schedule of the instrument. The acquisition of results is done by each user except for assisted sessions. During working hours (9-18H), the platform staff are available to the users to intervene in case of technical problems or malfunction of the instruments only. If the platform personnel are not present, call the manufacturer's technical support (label with telephone number and machine reference on the computer side of the cytometer cover). Any request for advice or assistance for the passing of samples is only made by appointment in the context of assisted sessions by clicking Request Assistance in the schedule of the instrument while booking your slot. The cytometry platform provides the sheath fluid for the cytometers. Other consumables, especially tubes, are brought in by each user. No samples should be present in the laboratory outside of the time taken to obtain the results. It is strictly forbidden to disassemble or modify the cytometers. 1/12/2017 version n 15 4

5 What to do before leaving the cytometer: Canto Canto II Fortessa The following hour is Clean flow cell on If you are the last If you are the last user of the booked Tube A, B, C user of the day of the day of the HTS module HTS module make a HTS clean. Clean The following hour is not booked but there are users later in the day Last user of the day leave everything on Clean flow cell on tube A, B, C leave everything on, switch off the cytometer and close the compressed air Clean flow cell on tube A, B, C Fluidics shut down. windows, switch off computer, cytometer and close the compressed air flow cell on Tube A, B, C leave everything on If you are the last user of the day of the HTS module make a HTS clean. Clean flow cell on Tube A, B, C leave everything on, switch off the cytometer and close the compressed air If you are the last user of the day of the HTS module make a HTS clean. Clean flow cell on Tube A, B, C Fluidics shut down and Log out and log off windows, switch off computer, cytometer and close the compressed air make a HTS clean. Run the Tube A, B and C on Run High for 3 min, leave the tube C and press standby. windows. Leave everything on If you are the last user of the day of the HTS module make a HTS clean. Run the Tube A, B and C on Run High for 3 min, leave the tube C and press standby. windows. switch off the cytometer If you are the last user of the day of the HTS module make a HTS clean. Run the Tube A, B and C on Run High for 3 min, leave the tube C and press standby. windows. switch off the cytometer The platform staff fills the "sheath, surfactant and clean" cans. Quality control and instrument calibration is performed once a week. Finally, every two weeks the instruments undergo a long cleaning cycle. 6 Data storage and analysis of results Cytometers are restricted to data acquisition only. The data generated by users are their responsibility and must not be stored in the software. They are exported to your personal folder or extranet folder on the server. The transfer must take place during the reservation period because the data is deleted from DIVA after 24 hours. A daily backup of the 1/12/2017 version n 15 5

6 server is done every night and your data is secured. However, for users who do not belong to the UMR5164, you must safely archive them on another media in your laboratory. To repatriate your data on another computer, the use of a USB stick or external disk (viral risk) is strictly prohibited. Use server sharing when you are in cytometry or at UMR5164 or Pydio when you are away from the service. Simply log in using the login and password provided to you. It is forbidden to reserve the machines for analysis, 4 computers are provided for this purpose. Their reservation is free of charge and is done like cytometers. 7 Mandatory health and safety rules to be applied Samples containing human cells or blood must be fixed (formaldehyde 1%) for analysis and any sample with mycoplasma or biological risk greater than level 2 (cells handled at L3) for the manipulator will be rejected for cell sorting. Samples presenting any danger or requiring special treatment for safety reasons must be subject to special authorisation by the platform operator. The cell sorters are located in a type 2 (L2) biosecurity laboratory. To get in, you must wear one of the green coats available on the coat racks. Gloves are available near the instruments and must be worn when passing samples. Gloves and all waste are disposed of in the yellow bio-waste bin before leaving the premises. Benches and machines must be left perfectly clean and free from any material belonging to you for the next user. Everything that is forgotten is put in the garbage. Spray a little antiseptic on the bench where you worked then wipe with absorbent paper. 8 Invoicing 2018 Analysers Sorters Canto 10 /h Canto II 15 /h Fortessa 20 /h *above 100H per semester, extra hours are charged 50 % Aria 8C 30 /h Aria PSM 35 /h The first bill for consumption from November to April is payable in May, the second bill for consumption from May to October is payable in November. Default invoicing is by group, if you want invoicing by laboratory/unit please select the option in your PPMS account request. 9 PPMS account creation request and agreement Please fill in and sign the form 1/12/2017 version n 15 6

Standard Operating Procedure Of The CytoFLEX S Flow Cytometer At The Institute Of Virology

Standard Operating Procedure Of The CytoFLEX S Flow Cytometer At The Institute Of Virology Before starting with the CytoFLEX S Flow Cytometer: 1. A new user needs to get an introduction to the flow cytometer