IGC

Analysers

We have currently 4 analysers available, each with different assets. The analysers are operated by the users themselves after a theoretical and a hands-on training. To request a training, please contact the IGC-FCF staff. The use of the analysers is paid per hour of reservation.

Before using an analyser, please read carefully the facility policies, in particular the policies for analysers.

Which analyser should I use?

To choose the cell analyser that best suits your experiment, check the optical configuration of each equipment and verified applications. You should ask yourself:

  1. How many colors do I need to read?
  2. Which instrument has the lasers suitable to excite all my fluorochromes?
  3. Which instrument has the detectors suitable to read the fluorescence emitted by my fluorochromes?
  4. Has someone already done something similar to my experiment? Which instrument did they use?

If you need advice or have any question regarding our analysers, contact the FCF staff.


List of Analysers and their configurations


BD Fortessa X-20

The X-20 is equipped with 4 different lasers. It can detect up to 16 colors simultaneously. Instead of calling the detection channels generically FL1-to-FL16, each channel is named after it's respective PMT detector, to easily setup an experiment users should label which fluorochromes are being detected. You can find an "How to use" guide by the side of the instrument or can download it here.

The software used for this machine is FACSDiVa (version). Know more about the software used here.

Cleaning Procedures

At the end of acquisition, it is mandatory to clean the flow cytometer. All cleaning solutions can be found in the cytometers room. For Fortessa X-20, the cleaning procedures are as follows:

Regular Cleaning Procedure

For cells labelled with surface or intracellular antibodies, or transgenic fluorescent proteins (without PI or other viability dyes)

  1. Prepare a tube of Bleach and place it on the SIP with the arm open for 60 seconds.
  2. Close the arm, and run and record 5 minutes of Bleach in SUPER HIGH.
  3. Repeat step 1 and 2 using a tube of Detergent, and a tube of H2O. Don't forget to save the cleaning files naming them Detergent and H2O, respectively.
  4. Ensure that after 5 minutes of H2O, there are no more than 300 events recorded.
  5. If more than 300 events were detected in the last step, please follow these indications:
    1. Prepare a new tube of H2O and check if the number of events has decreased;
    2. Prepare a new tube from a different H2O falcon and check if the number of events has decreased;
    3. Prime the machine 3 times;
    4. Repeat the entire cycle of cleaning procedures.
  6. If there is less than 300 events, check if there are users after you and decide whether to turn OFF the equipment or leave it ON in Standby.

Cleaning procedure for cells labeled with PI or other viability or DNA dyes

Option 1: At the end of your experiment, acquire a sample of very concentrated unstained cells (e.g. cultured cells, spleen, thymus, etc.) for 3 to 5 minutes. Record this tube naming it “Unstained to clean PI”. Then perform the Regular Cleaning Procedure.
Option 2: Perform the regular cleaning protocol, but extend the time of detergent running step to 10 minutes.



BD LSR Fortessa

The BD LSR Fortessa is equipped with 3 different lasers, a FSC-PMT and is coupled to a High Throughput Sampler (HTS) that allows the acquisition of samples in 96-well plates. Due to the FSC-PMT, this equipment is particularly suitable for the detection of small cells or particles, such as bacteria. The optical configuration for detection must be set by each user upon selection of the optical filters and dichroic mirrors to detect the target fluorescences (see list of available filters and mirrors below). You can find an "How to use" guide by the side of the instrument or can download it here.

The software used for this machine is FACSDiVa (version 6.2). Know more about the software used here.

Base Configuration (pdf)
YFP-CFP-DsRed-E2Crimson-E-coli (pdf)
YFP-CFP-orangish-E-coli (pdf)
GFP YFP CFP DsRed mCherry (pdf)
PI 7AAD PE GFP (pdf)
TFP GFP sYFP mCherry E coli (pdf)
YFP CFP mCherry E coli (pdf)
YFP CFP mCherry E coli (v2) (pdf)
GFP mCherry SPombe (pdf)
GFP YFP RFP DBodor (pdf)
GFP PI CFP RFP (pdf)
YFP CFP BacLight E coli (pdf)
YFP CFP E coli (pdf)
CY3 YFP CFP E coli (pdf)
GFP RFP (pdf)
GFP CFP RFP (pdf)
PE PerCp PE-Cy7 (pdf)
PE PE-Cy55 PE-Cy7 (pdf)
Af594 PeCy5 (Bacteria) (pdf)


Cleaning Procedures

At the end of acquisition, it is mandatory to clean the flow cytometer. All cleaning solutions can be found in the cytometers room. In Fortessa, the cleaning procedures depend on whether the acquisition was performed in tubes or in plate.

Cleaning procedure for acquisition in tubes:

Regular Cleaning Procedure

For cells labelled with surface or intracellular antibodies, or transgenic fluorescent proteins (without PI or other viability dyes)

  1. Prepare a tube of Bleach and place it on the SIP with the arm open for 30 seconds.
  2. Close the arm, and run and record 5 minutes of Bleach in SUPER HIGH.
  3. Repeat step 1 and 2 using a tube of Detergent, and a tube of H2O. Don't forget to save the cleaning files naming them Detergent and H2O, respectively.
  4. Ensure that after 5 minutes of H2O, there are no more than 300 events recorded.
  5. If more than 300 events were detected in the last step, please follow these indications:
    1. Prepare a new tube of H2O and check if the number of events has decreased;
    2. Prepare a new tube from a different H2O falcon and check if the number of events has decreased;
    3. Prime the machine 3 times;
    4. Repeat the entire cycle of cleaning procedures.
  6. If there is less than 300 events, check if there are users after you and decide whether to turn OFF the equipment or leave it ON in Standby.

Cleaning procedure for cells labeled with PI or other viability or DNA dyes

Option 1: At the end of your experiment, acquire a sample of very concentrated unstained cells (e.g. cultured cells, spleen, thymus, etc.) for 3 to 5 minutes. Record this tube naming it “Unstained to clean PI”. Then perform the Regular Cleaning Procedure.
Option 2: Perform the regular cleaning protocol, but extend the time of detergent running step to 10 minutes.


Cleaning procedure for acquisition in plates:

Record the acquisition of one line of a 96-well plate as follows:

  1. 3 wells with 300 µL bleach (name wells 'Bleach')
  2. 3 wells with 300 µL bleach (name wells 'H2O')
  3. 3 wells with 300 µL bleach (name wells 'Detergent')
  4. 3 wells with 300 µL bleach (name wells 'H2O')

Run the selected wells in Standard mode with the following settings:

After the cleaning procedure has completed, check if there are users after you and decide whether to turn OFF the equipment or leave it ON in Standby.


BD FACSCalibur

FACSCalibur is equipped with 2 lasers and can read 4 colors simultaneously. It is a very good cytometer to run cell cycle assays using propidium iodide (PI). You can find an "How to use" guide by the side of the instrument or can download it here.

The software used for this machine is CellQuest Pro 6.0. Know more about the software used here.

Cleaning Procedures

At the end of acquisition, it is mandatory to clean the flow cytometer. All cleaning solutions can be found in the cytometers room. For FACSCalibur, the cleaning procedures are as follows:

Regular Cleaning Procedure

For cells labelled with surface or intracellular antibodies, or transgenic fluorescent proteins (without PI or other viability dyes)

  1. Prepare a tube of Bleach and place it on the SIP with the arm open for 30 seconds.
  2. Close the arm, and run and record 5 minutes of Bleach in HIGH.
  3. Repeat step 1 and 2 using a tube of Detergent, and a tube of H2O. Don't forget to save the cleaning files naming them Detergent and H2O, respectively, in the Sample ID field.
  4. Ensure that after 5 minutes of H2O, there are no more than 300 events recorded.
  5. If more than 300 events were detected in the last step, please follow these indications:
    1. Prepare a new tube of H2O and check if the number of events has decreased;
    2. Prepare a new tube from a different H2O falcon and check if the number of events has decreased;
    3. Prime the machine 3 times;
    4. Repeat the entire cycle of cleaning procedures.
  6. If there is less than 300 events, check if there are users after you and decide whether to turn OFF the equipment or leave it ON in Standby.

Cleaning procedure for cells labeled with PI or other viability or DNA dyes

Option 1: At the end of your experiment, acquire a sample of very concentrated unstained cells (e.g. cultured cells, spleen, thymus, etc.) for 3 to 5 minutes. Record this tube naming it “Unstained to clean PI”. Then perform the Regular Cleaning Procedure.
Option 2: Perform the regular cleaning protocol, but extend the time of detergent running step to 10 minutes.


CyAn ADP

CyAn ADP is equipped with 3 lasers and enables the detection of at least eight colors. The software used for this machine is Summit.

Cleaning Procedures

At the end of acquisition, it is mandatory to clean the flow cytometer. All cleaning solutions can be found in the cytometers room. For CyAn, the cleaning procedures are as follows:

Regular cleaning procedure

For cells labelled with surface or intracellular antibodies, or transgenic fluorescent proteins (without PI or other viability dyes)

  1. Prepare a tube of Bleach and run it at the highest flow rate for 5 minutes. Record this tube naming it “Bleach”.
  2. Repeat step 1 with a tube of Detergent, and a tube of H2O. Don't forget to save the cleaning files naming them Detergent and H2O, respectively.
  3. Ensure that after 5 minutes of H2O, there are no more than 300 events recorded.
  4. If more than 300 events were detected in the last step, please follow these indications:
    1. Prepare a new tube of H2O and check if the number of events has decreased;
    2. Prepare a new tube from a different H2O falcon and check if the number of events has decreased;
    3. Press debubble 6 times;
    4. Repeat the entire cycle of cleaning procedures.
  5. If there is less than 300 events, check if there are users after you and decide whether to turn OFF the equipment or leave it ON in Standby..

Cleaning procedure for cells labeled with PI or other viability or DNA dyes

Option 1: At the end of your experiment, acquire a sample of very concentrated unstained cells (e.g. cultured cells, spleen, thymus, etc.) for 3 to 5 minutes. Record this tube naming it “Unstained to clean PI”. Then perform the Regular Cleaning Procedure.
Option 2: Perform the regular cleaning protocol, but extend the time of detergent running step to 10 minutes.

Pricing

Type
Price/hour
Internal
11.14 €
Academic
11.14 €
Campus
11.14 €
Industry
23 €

General Policies For the Usage of Analysers

  1. It is mandatory to have a reservation in Agendo to use any cytometer.
  2. Before collecting samples, make sure the cytometer is clean by running (and recording) 1 tube of H2O for 1-3 minutes. The cytometer is clean if there are less than 200 events in 3 minutes (approx. 1 evt/s).
  3. It is mandatory to filter samples before analysis in the cytometer to prevent clogs. The FCF staff can provide sterile re-usable filters when needed.
  4. Users are responsible to clean the analysers after use, following the cleaning procedures posted on the instruments and described on the analysers tab above.
  5. Users must record the data of the cleaning steps, to enable the facility staff to track the origin of a potential problem.
  6. After using the cytometer, investigators are responsible for leaving the cytometer room clean. This includes: placing the solution flasks and pipettes back in their dedicated bench, discarding unused tubes, trashing used paper tissues and used tips.
  7. Users are responsible for exporting and saving their data.
  8. Users must refill the PBS tank after using the analyser.
  9. The users with the last reservations of the day must ensure the equipment is turned OFF, even if they don't show up to the reservation.
More information regarding facility policies here.