Flow Cytometry and Antibodies

The Flow Cytometry & Antibodies Unit offers flow cytometry and antibodies-related services and expertise to IGC researchers, as well as to outside groups and companies.

Laboratory staff is well trained in instrument operation, providing support to cell sorting, cell acquisition and data analysis. Full service is provided for antibodies-related requests and include production of monoclonal antibodies, antibody purification and fluorescent labeling of antibodies and proteins. Training is available for all analytical instruments and SOP are implemented to comply with the highest quality standards required to ensure reproducibility in science.

The need to find solutions to support research projects drives a continuous development of the facility, which aims to follow the advances in the flow cytometry field, collaborates with innovative projects, creates novel tools and methods to advance research, and implements strategies to improve the quality of the provided services.

For any questions regarding flow cytometry and antibodies services please contact [email protected].

 

Users:

1. The IGC-FCA is open to IGC researchers and to investigators outside IGC.
2. New users must first register in our booking system, Agendo, and then request permission to gain access to the calendar of the desired equipment.
3. To use the FCA services, users aiming to do flow cytometry analysis must enroll in our Training Program, consisting of one theoretical and one hands-on module. This training is optional for those users only doing cell sorting.
4. Users may be charged for assistance if needed.
5. All users must read, understand and agree with the policies described here. Any questions should be directed to the IGC-FCA staff.


Reservations:

1. It is mandatory to reserve through Agendo any equipment before usage, including weekends.
2. To gain access to instrument calendars, users must request permission for the desired equipment through Agendo.
3. Using equipment without reservations is forbidden and will be reported to the Head of FCA and to the IGC Direction as misconduct.
4. An instrument can be booked up to 14 days in advance in blocks of 30 minutes (analyzers) or one hour (sorters).
5. Booking of consecutive slots of time for the same user is not allowed. Users must make a single reservation for the entire time period intended.
6. Experiments are scheduled on a first-booked-first-served basis.
7. Filling all reservation fields is mandatory, and must be in accordance with the field's title. All requested information is necessary for assistance and/or statistical purposes, to help improve the quality of the service. Failure to fill adequately the requested fields will be considered improper usage and may lead to reserving limitations for the abusing user.
8. Reservations for analyzers are automatically confirmed upon user login in Agendo on the workstation connected to the respective equipment, within a specified period of time before, during, or after the reservation time. Failure to do so indicates the user did not use his/her reservation slot, being automatically reported by e-mail to the user and to the FCA staff.
9. All the reservations are charged, unless they are deleted.
10. If the user decides not to use the equipment, the reservation must be deleted at least four hours (analyzers) or 48 hours (sorters) in advance.
11. When deleting a last reservation of the day, investigators must ensure that the previous user and the FCA staff are both notified.
12. Users must be aware that even if their reservation is not the last one of the day, it may become so if subsequent slots of other users are deleted meanwhile. Therefore, every user must check the Agendo calendar before leaving the analyzer’s room, to ensure he/she has not become the last user of the day.
13. The full responsibility for the proper usage of the equipment belongs to the user who has the reservation in his/her name.
14. Reservations for the cell sorters work as pre-reservations and require confirmation from the FCA facility according to staff availability.
15. Filling all reservation fields for the sorting pre-reservation is mandatory to help FCA staff do determine the best instrument, configuration, settings and sorting time.
16. Approval of and/or changes to the pre-reservation will be notified by e-mail.
17. It is the responsibility of the PIs to ensure their students and postdocs comply with all the policies described here.


Resources:

1. Cell Sorters – FACSAria IIu

a. The instrument and operators are available for sorts Monday through Friday, from 10h00 to 18h00, except during maintenance periods (see Section Weekly Maintenance below).
b. The sorts are scheduled on a first-booked-first-served basis.
c. Cell sorting scheduling is done at the discretion of the FCA, to maximize availability for the entire IGC community. FCA staff schedules sorts preferentially from the beginning or at the end of the day to avoid breaking large time blocks.
d. If time is unscheduled one day prior to sort, FCA staff can adjust the schedule to fit better particular user needs.
e. To schedule a sort, please:
   i) Check time availability for the intended instrument in Agendo
   ii) Fill up all the fields required upon Agendo booking
   iii) Get follow-up confirmation e-mail from FCA staff
f. Prior to the first sort of a new protocol, please arrange an appointment with FCA Head to discuss the details of the sorting experiment.
g. All the sorting experiments are performed by FCA operators, unless users choose to attend the FCA training module of Cell Sorting and acquire sufficient experience to become ‘Power Users’.


2. Flow Cytometer Analyzers – FACSCalibur (on BSL-3), LSR Fortessa SORP, LSR Fortessa X20, Aurora Cytek

a. The analyzers are available 24/7, except during maintenance periods (see Section Weekly Maintenance below).
b. Data acquisition on the Flow Cytometry cell analyzers is performed by investigators in a self-service regimen.
c. To operate flow cytometer analyzers, investigators must attend first the FCA Training Program, consisting of one theoretical lesson and one practical session at the instrument.
d. All the experiments are performed autonomously by researchers, unless FCA staff assistance is requested.
e. To get priority assistance, please make sure to schedule it in advance by contacting the FCA staff.
f. For charges of FCA staff assistance, please refer to Charges and Fees.
g. It is mandatory to decontaminate the flow cytometer analyzers immediately after sample acquisition, following the cleaning procedures indicated by the FCA. Detailed notes on washing procedures are posted on the instruments.
h. After decontamination, the cytometer must be switched to STAND BY and stay with a tube containing distilled H2O installed.
i. It is mandatory to refill the PBS container at the end of acquisition.
j. Instruments, computers and screens should be turned off if nobody is scheduled to use it in the following four hours and after the last user of the day.
k. The last user of the day is fully responsible for shutting down the equipment. Failure to do so may incur in penalties for the user and costs for the PI for any damage directly related to this serious misusage.
l. It is strictly forbidden to remove the Sample Injection Port (SIP) outer sleeve from any instrument. If you believe you need to do so, please contact the FCA staff.
m. If the user experiences any unusual behavior with the cytometer and/or during the analysis, he/she should first refer to the Troubleshooting section of the User’s Guide that is available next to each instrument.
n. If the situation cannot be solved according to the Troubleshooting recommendations, the user should call the facility's staff for assistance.
o. It is the user’s responsibility cleaning up the working bench space and discard all the tubes into the green containers for biohazard waste after his/her experiment.


3. Analysis Workstation – Tango Data Analysis Workstation

a. The computer is available 24/7.
b. Access to the computers is on first-come-first-served basis.
c. These computers are dedicated to flow cytometry data analysis only. They are on the IGC network, run Flow Cytometry software (FlowJo, FCS Express, SpectroFlo, ModFit, Milliplex Analyst, IDEAS) and Office programs to analyze and publish data.
d. The local hard disks of the analysis workstations are not for long-term data storage.
e. Users are responsible to make archival copies of their data.


Data Management

1. The local hard disks of the cytometer workstations and Tango analysis workstaion are not for long-term data storage.
2. All files are routinely (monthly) erased to maintain enough free disk space for acquisition and/or analysis, including data on FACS Diva and SpectroFlo.
3. It is strongly recommended to export experiments from the FACS Diva and SpectroFlo to the local workstation folder immediately after acquisition, and transfer data from there to the user’s personal directory in the remote server.
4. The local folder that should be used to store temporarily data exported from FACS Diva and SpectroFlo is located in the Desktop (Export – Routinely Cleared). Any files or folders outside this folder will be deleted without notice.
5. The use of USB pens or external drives is not allowed.
6. The IGC-FCA is not liable for any loss of data on the local hard disks.

 

Weekly Maintenance

1. Regular weekly maintenance on all FCA cell sorters and analyzers is scheduled on Monday or Tuesday mornings (see time scheduled in Agendo).
2. Regular weekly maintenance includes the following procedures on all FCA Analyzers:
a. Fluidic system flush according to manufacturer-recommended procedures for Weekly or Monthly Maintenance (analyzers); extended acquisition of cleaning solutions & preparation for aseptic sorting (FACSAria);
b. Autoclaving of FACSAria sheath tank (twice a month);
c. Quality control according to manufacturer-recommended procedures (all instruments);
d. Reposition of all cleaning solutions, PBS and dH2O, and 5-mL FACS tubes;
e. Removal and discard of all unlabeled sample tubes and racks from the desks and benches;
f. Purge of all illegitimate files from the computers (at the beginning of every month).

 

Charges and Cancellation Policy

1. To recover essential operating costs, the FCA charges hourly fees for the use of the all analytical instruments.
2. A fee for each FlowJo license is charged annually to groups requesting these licenses for their personal computers.
3. Usage of cell counters is free of charge, but scepter sensors needed for cell counting are charge.
4. Usage of analysis workstations (other than those attached to the instruments) is free of charge.
5. Scheduled operator-assisted services (sorting and MAGPIX), as well as BSL-3 cell analysis, must be canceled no later than 48 hours prior to the scheduled experiment. If the scheduled sort is not cancelled by this time, the booked time will be fully charged, even if the investigator decides not to use it.
6. Scheduled use of BSL-1cell analyzers must be cancelled no later than four hours in advance of the scheduled start time. If the scheduled use of the analyzer is not cancelled by this time, the booked time will be fully charged, even if the investigator decides not to use it.
7. If investigators do not cancel their booking within the free-of-charge tolerance period, but find someone willing to use the time they had booked, the FCA staff will transfer that booking to the name of the second investigator, so it is charged to the new user.


Training, Consultation, Discussion and Troubleshooting

1. The subjects reviewed at the training sessions may vary depending on the background, needs and interests of the investigators. Please do not hesitate to contact FCA Head to find out the best solution for you.
2.The Theoretical Introduction sessions are scheduled on a regular basis; the Practical Training sessions are scheduled with the FCA staff on a first-booked-first-served basis after attending the Theoretical Introduction module.
3. For consultation and help, please contact directly the FCA staff during regular working hours or 24/7 via e-mail to get immediate feedback or schedule a meeting with the head of the facility.


Publication and Acknowledgment of the Facility

1. By using the IGC Flow Cytometry & Antibodies Facility services, investigators commit to refer the Facility in all publications that include data generated by its services and/or resources in the Acknowledgements section. You are welcome to use the following examples of references: “The authors gratefully acknowledge the IGC Flow Cytometry & Antibodies Facility for their support and assistance with this work”.


EQUIPMENT

BD Fortessa X-20
Analyser BD LSR Fortessa SORP
Analyser BD FACS Calibur
Analyser CYTEK Aurora
Sorter BD FACS ARIA IIu
Cell Counter Scepter Millipore
Multiplex Reader MAGPIX
Purification System AKTA Pure
Cell Metabolic Analysis Seahorse XFe96 Analyzer
Data Analysis Resources Tango Data Analysis Workstation

SERVICES

The facility provides a limited number of one-year licenses for FlowJo, a flow cytometry data analysis software with user-friendly environment.

Flow Cytometry & Antibodies Facility offers high quality training in flow cytometry, including cell sorting, full spectrum acquisition and data analysis, providing lectures and hands-on sessions.

The Flow Cytometry & Antibodies Facility has integrated quality assurance protocols for the operation of all scientific instruments.

Small to medium scale monoclonal antibody production from hybridomas (10 mg to 100 mg) provided in full service.

Selection and expansion of the most efficient hybridoma sub-clones for maximizing antibody production.

Custom conjugation of antibodies or proteins with specific organic fluorescent dyes, followed by purification, quantification and validation of the reagents for flow cytometry or other applications.

Highly sensitive polymerase chain reaction (PCR)-based service to reliably detect myscoplasma in cell cultures, with results in 3-5 business days.

Cell culture service to provide: cell line maintenance and/or scale-up; ready-to-use cells; cell culture quality control; cell line banking and storage.

Design and optimization of antibody-fluorochrome combinations to build and validate large multicolor panels for flow cytometry.

Incubation of samples with titrated fluorescent dyes and/or labeled antibodies for flow cytometry.

Advise on projects, experimental approaches, data analysis and presentation regarding Flow Cytometry and/or antibody related experiments.


USEFUL RESOURCES


PROTOCOLS


PEOPLE


PUBLICATIONS

Check the papers here.


MEDIA


 

Funding                                                                                                                                            Consortiums

                                                                                                                                                             

Updated on 10 may 2022

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