Genetic complexity of living systems

FACSCalibur Becton Dickinson HTS

Startup procedures :

-If everything is OFF: Start up FIRST the cytometer and the pump (under the bench) and THEN the computer (if you need the user and login, please ask us). The cytometer will indicate « not ready » during about 10 minutes. If the computer is ON you MUST restart it.

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

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

-If the cytometer is on Standby and without pressure (cursor behind= pressure OFF)

Go to the HTS mode:

-1: Switch  the big button from “tube” to “plate” (behind the needle)

-2: Change the SURneedle (put the shortest one, on the right door of the cytometer)

            *pull the lever

            *unscrew the SURneedle (be careful: the needle is more flexible)

-3: Attach the “sample coupler” on the needle

-4: Switch on the HTS button (on the right)

-Close the cover to start

-“Cell Quest Pro” must be closed to start HTS (or to start an new acquisition), open “Plate Manager”

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

-Close the cover

-Run on the cytometer

-Go to sampler and make 2 “prime”

-Go to : File/New from template/HD1/LBMC/Yvert/Settings/HTS and choose TemplateHTS96puits.

-Go to: Layout to configure the plate

*Select the mode: Standard

*Select the plate: BD-96-flat

-Go to: Sampler parameters:

*data file prefixe part #1: date of the day

*data file prefixe part #2: name of the experiment

*data file prefixe part #3: more informations if necessary

*acquisition document: HD1/LBMC/Yvert/noise_acquisitionHTS (10000events/s)

*analysis document:

*instrument settings: HD1/LBMC/Yvert/Settings_noise_acquisition_2

*Sample volume: 100 uL

*Sample rate: 1 ul/s

*Mixing volume: 100 uL

*Mixing speed: 180 uL/s

*Probe wash volume: 400uL

*Number of mixes: 4

Select the wells you want to acquire and clic on samples (numbers will appear on each samples)

Acquisition of samples :

-In a 96well plate, put 200uL TBS1x and a some uL (6 to 8 uL) of the culture (resuspend cells before pipetting) to have 400-500 events/s.

-Put the 96well plate in place: A1 must be on the right top corner.

-Set up: if you want to acquire without recording

-Acquire: acquisition and record. Choose a folder with the date of the day and a name for the file.

When it’s OK Start the acquisition.

You're not done! Now comes the cleaning step:

-In the cleaning plate put 250uL per well of rinse in 2 rows and 250uL per well of water in 2 next rows.

-Replace the experimental plate by the cleaning plate, close the cover.

-Go to: File/New from template/HD1/LBMC/Yvert/Settings/HTS and choose TemplateHTS_CLEAN

-Go to: Layout to configure the plate

*Select the mode: Standard

*Select the plate: BD-96-flat or U

*Sample volume: 200 uL

*Sample rate: 3 ul/s

*Mixing volume: 10 uL

*Mixing speed: 180 uL/s

*Probe wash volume: 400uL

*Number of mixes: 2

Select the wells

-Go to: Acquire: acquisition and record. Choose the "clean" folder.

When it’s OK Start the acquisition.

It take about one hour.

When it’s finished:

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

-1: Switch OFF the HTS button (on the right)

-2: Remove the “sample coupler” from the needle

-3: Change the SURneedle

-4: Switch  the big button from “plate” to “tube” (behind the needle)

-Quit Plate Manager, don’t save

You're not done! Now comes the control of the efficency of the cleaning step:

-Open "Cell quest pro".

-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-1/Open/Set/Done

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

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

-Run on the cytometer

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

-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)).

-Transfer the results in your computer with a USB key.

-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 !

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

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

            *the date

            *first and last name

            *unit and team : LBMC-GY

            *cellular type : yeast/levure

            *Beginning time(*)

            *Ending time (*)

            *Number of plate

(* : 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.