Cytometry

• All users must fill out the biosafety form before working with potentially infectious materials or planning a cell sorting experiments. 
• Users must always wear gloves and a lab coat when using the cytometers or their associated computers.
• It is strictly forbidden to drink or eat in the core facility premises.
• Users who intend to analyze infectious materials of human nature (bacteria, viruses, human or primate cells) must fix the cells (usually with 2-5% paraformaldehyde or 70% ethanol).
• Potentially infectious samples brought to the core facility must be secured (sealed cap, closed transport box) and clearly labeled. The core facility has the right to refuse to analyze samples that do not comply with biosafety standards.
• In case of accidental spillage, it is mandatory to disinfect the contaminated surface according to the appropriate protocol.
• Cleaning workstations with 70% ethanol is mandatory at the end of each appointment.

• Only users who have undergone training can use the analyzers independently. Tailored training is available. For more information, click on the “Training” tab.
• Reservations are made through the TeamUp calendar on a “first come, first served” basis and subject to cytometers availability.
• You are responsible of the cytometer for the entire duration of your reservation. Cytometers should NEVER be left unattended unless in “STANDBY” mode. Leaving a cytometer in “RUN” mode for too long can damage it and incur significant costs.
• After 4:00 PM, on weekends, and on holidays, cytometers must be shut down after each use unless the next user is physically present or an agreement has been made with them.
• As a courtesy to other users, you are required to respect your appointment time.

• Analysis with technical assistance is by appointment only, subject to operator availability.
• It is important to provide a summary of your experiment when scheduling an appointment in order to choose the instrument best suited to your needs.
• Analysis with technical assistance is available from 9:00 AM to 5:00 PM, Monday to Friday (excluding holidays). For analysis at other times, please contact us to plan.
• All cancellations with less than 6 hours’ notice will be charged as follows: 30 minutes plus 25% of the remaining reserved time slot.
• In case of unjustified absence, the entire reserved appointment will be charged.

Sample Preparation for Analysis

• Fluorochromes used should always be properly titrated before planning a complete experiment. Titration saves time, money and improves the quality of your results.
• Suspend samples in PBS at a concentration not exceeding 5×106 cells/ml. Bovine Serum Albumin (BSA) should not be used as it increases the risk of clogging the cytometer. Serum is a good alternative.
• It is not mandatory to filter samples for analysis. However, to facilitate the process and reduce analysis time, it is recommended to filter samples forming aggregates with a 30 to 50 µm filter.
• Samples should be kept on ice (or in the refrigerator) protected from light to avoid altering cellular phenotype or fluorochromes.
• Control samples: For all analysis, an unstained control as well as single-stained controls are required for the instrument calibration. For example, if using FITC, PE, and APC, you need four controls in addition to the samples: unstained + one single-stained control for each color. FMO (Fluorescence Minus One) controls are also recommended in some cases. Additional controls may be required depending on your experiment or the instrument used.
• Fluorescent proteins (e.g., GFP, mCherry, YFP): Fluorescent proteins should be considered as regular fluorochromes. An untransfected control and a control expressing the protein are required for each experiment.
• Controls should always be prepared under the same conditions as the samples.

Before leaving, all users must follow the following cleaning protocol: 

1. Run 10% bleach for 5 minutes. 
2. Run water for 5 minutes. 
3. Put the cytometer in “STANDBY” mode and set the speed to “LOW” (Symphony A1).
4. Close your session in FACS Diva. 
5. Refill the sheath and empty the waste if needed. 

When you are the last user of the day, you must also: 

1. Shut down the cytometer. 
2. Shut down the computer. 

Fresh solutions are always available in the lab. 

Users who are negligent may lose their access rights to the core facility without notice. All costs incurred by the core facility due to negligent use will be charged in full to the principal investigator responsible of the user. 

• Cell sorting is by appointment only. It is important to clearly explain your needs with as much detail as possible when scheduling an appointment.
• For a new experiment, the biosafety form must be filled out for your appointment to be approved.
• Sorting appointments start between 10:00 AM and 2:00 PM, Monday to Friday. An alternative time can be discussed when scheduling the appointment.
• For any cancellation within 24 hours, 1 hour of time plus 25% of the remaining reservation slot will be charged. For an unjustified absence, the entire reserved time slot will be charged.
• It is recommended to book at least 1 week in advance. Appointments are granted on a first-come, first-served basis.
• It is highly recommended to perform at least one analysis before cell sorting to prepare an adequate number of cells.

Sample Preparation

• Do not fill more than 2/3 of the tube. If necessary, split the sample into multiple tubes.
• Suspend samples in PBS at a concentration of 5×106 to 1×107 cells per ml. Bovine Serum Albumin (BSA) should not be used as it increases the risk of clogging the cytometer. Serum is a good alternative.
• Cell concentration affects sorting speed and efficiency. Use the highest possible concentration (while maintaining cell suspension). It is possible to dilute a sample, but not to concentrate it.
• Samples must be filtered (30 to 50 µm filter) before sorting to avoid aggregates or doublets. Aggregates affect sorting speed and efficiency.
• Control samples: For all sorts, an unstained control as well as single-stained controls are required for the instrument calibration. For example, if using FITC, PE, and APC, you need four controls in addition to the samples: unstained + one single-stained control for each color. FMO (Fluorescence Minus One) controls are also recommended under certain conditions.
• Fluorescent proteins (e.g., GFP, mCherry, YFP): Fluorescent proteins should be considered as regular fluorochromes. An untransfected control and a control expressing the protein is required for each experiment.
• Controls should always be prepared in the same way as the samples.

Preparation of the Collection Tubes

• Pre-coating tubes with serum or medium with serum is recommended to improve sorting efficiency.
• Medium with or without serum should be added to the collection tubes (300 µl in 12x75mm tubes and 2-3 ml in 15 ml tubes).

Sorting into Plates

• It is possible to sort from 1 to 50,000 cells into 6 to 96-well plates.
• Plates should be filled with medium (100 µl in 96-well plates).

For any questions, feel free to contact the core facility (cytometry@crchudequebec.ulaval.ca) to discuss the design of your experiment. 

It is STRICTLY PROHIBITED to use personal USB drives or external hard drives to transfer your files. These may contain viruses and affect the proper functioning of the cytometers. 

The flow cytometry core facility will keep your data from recent experiments. However, it is your responsibility to make a backup copy of your data in a safe location outside the core facility (cloud, USB drive, DVD, server, computer).

CHU de Québec-Université Laval users must use the specific TeamUp link of their team to make a booking. If your team has not received a link, feel free to contact us to create one. Each link allows all members of a team to make reservations independently.

Cytometer Booking Procedure

If you wish to use the core facility’s services, please contact us at cytometrie@crchudequebec.ulaval.ca so that we can plan your project with you.

Technical assistance at the core facility is available Monday to Friday from 9:00 a.m. to 5:00 p.m. (excluding holidays). For special requests or last-minute appointments, you must contact the platform before booking your appointment to make sure someone is available. All appointments requested more than 15 days in advance may be modified if technical assistance is not available. All technical assistance hours outside normal office hours will be charged at time and a half. 

Internal users must make their reservations through the TeamUp link provided by the platform. External users must contact the platform to schedule an appointment (cytometrie@crchudequebec.ulaval.ca). It is strongly recommended to reserve your time slot at least 24 hours in advance for an analysis and at least 1 week in advance for cell sorting. If you book at the last minute, your appointment may not be accepted. All reservations are subject to platform approval. 

Analyzers

The minimum reservation time is 30 minutes. The maximum reservation period is 8 consecutive hours per person. A reservation for an analyzer canceled within 6 hours will be charged as follows: 30 minutes + 25% of the reserved time. If a user demonstrates negligence, they may lose their right to use the instruments. All costs incurred by a negligent user will be charged to the responsible researcher. 

Sorters

The minimum reservation time is 60 minutes. A reservation for a sorter canceled within 24 hours will be charged as follows: 1 hour + 25% of the reserved time. 

As a courtesy to other users, you should only reserve the time slot you intend to use. The billed time will be the greater between the time booked on the calendar and the actual time spent on the cytometer. It is possible to take more time on the cytometer if it is not booked by another person. However, a user who has booked on the calendar and is on time always has priority. In the case of unjustified absence, the booked time will be charged in full. Users who regularly arrive late or repeatedly cancel below the minimum times may be restricted from accessing the core facility.

To view the booking calendar for cytometers, click on the following link: https://teamup.com/ksgwyyzvmag3fqtdz1