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.