Genetic complexity of living systems

FACSCalibur Becton Dickinson

Startup procedures :

-If everything is OFF: Start up FIRST the cytometer and the pump (under the bench) and then the computer. The cytometer will indicate « not ready » during about 10 minutes.

-Check the level of FACSflow (sheath) and waste fluid containers. Change if necessary (Switch off the pump).

-When the waste fluid is full:  replace it by an empty Facsflow container add a pastille of Javel and 150uL of anti-foam (on the right door of the cytometer).

-When the cytometer is on Standby or Ready, put a new tube of water (be careful the bottle of water can be dirty)

-Put the pressure on (between the 2 containers : cursor in the front = pressure ON)

During the 10 minutes of washing in HIGH, on the computer :

-User : cytometrie and Password : cytometrie

-Open CellQuestPro.

-Go to : Acquire/Connect to cytometer

-Go to : File/Open Document/HD1/LBMC/Yvert/noise_acquisition

-Go to : Cytometer/Instrumentsettings/Open/HD1/LBMC/Yvert/Settings_noise_acquisition_2/Open/Set/Done

-Go to : Acquire/Parameters Description and Acquire/Counters

-In the open window of Parameters description go to Global Stettings/Global Acquisition & Storage Settings : verify that there are 15000 events and validate by pressing Enter.

-In the open window of Parameters description go to Directory/change/HD1/LBMC/Yvert/Results/Your name choose NewFolder and create one with the date of the day : ex : 20110224

-In File/change put the name of your samples

-Check that the cytometer is clean (no event in 30secondes), doing a start (but let the Set Up ticked) and then pause/abort

Acquisition of samples :

-Vortex the culture tube, make a dilution of samples in 1mL TBS1X (try 20µL in 1mL), vortex and put the tube on the FACSCalibur. Start, control if the number of events/seconds is<200.

If not, make a new dilution. When it’s OK Start the acquisition (the recording of the samples).

Pause/abort/ click on SetUp and Start.

-after the acquisition, click on SetUp, change the name of the file, dilute the next sample and do a new acquisition.

-If there is time between 2 samples, put a tube of water in LOW mode.

-Complete as one goes along the « Cytometer Experiment Data Sheet »

At the end :

-Run cleaning solution (CLEAN) for 10 minutes at high sample rate

-Run RINSE for 20 minutes (or 5min if someone is after*)

-Run Distilled water (new tube) for 20 minutes (or 5 min if someone is after*)

(* : if only one person is after you, make sure that he/she will come)

- Check the cytometer is clean (0event/second during 30 secondes)

-Leave machine on standby and put the pressure off (cursor at the back)

-Check the level of FACSflow (sheath) and waste fluid containers. Change if necessary (Switch off the pump).

(When the waste fluid is full:  replace it by an empty Facsflow container add a pastille of Javel and 150uL of anti-foam (on the right door of the cytometer)).

-Connect the results in your computer (Go/Connect to server…)

-Delete the results from the previous experiment (be sure, it’s been recorded in your computer before). Leave only one tempory file/person on the cytometer’s computer !

-Acquire/Disconnect from the cytometer

-Quit CellQuest Pro, don’t save

-If nobody after : put the computer, the cytometer and the pump OFF

-Note on the sheet « Utilisation du Facscan UMR 5239 »

            *the date

            *first and last name

            *unit and team : LBMC-GY

            *cellular type : yeast/levure

            *Beginning time(*)

            *Ending time (*)

(* : only the acquisition time, do not count the preparation time and the cleaning time)

In case of problem : contact Sandrine Mouradian (86-69) in Laurent Schaeffer ‘s lab.

Dernière mise à jour : ( 02-02-2011 )