SCF – Flow Cytometry January 2015
BD Fortessa HTS Procedure:
1) Remove the tube of water from the SIP and connect the HTS sample coupler.
Leave the support arm to the side.
2) Choose “New Experiment”.
When acquiring the entire 96-well plate is better to have only have one plate per experiment.
3) Click new plate and choose the plate type.
4) Use the Plate window to select the throughput mode (standard mode is recommended).
A maximum of 10µL of sample is acquired in High Throughput mode.
5) Plate Set-up:
i. Setup Controls (unstained cells) - recommended at least two wells of unstained cells to set all setting (no data is saved)
ii. Compensation wells – Select well > Experiment > Compensation setup >
Create compensation controls
iii. Specimen (samples) - Select the wells to acquire the samples 6) In the Plate window, adjust the Loader Settings for the wells.
Ensure each well contains sufficient volume + dead volume
7) Right click in the Acquisition window and choose “Show Plate Control”.
8) Make sure that you are set to record the right number of events from the desired gate.
9) Select the set-up control wells in the plate layout and click “RUN Well(s)”.
No data file is saved.
10) Adjust the settings to optimize FSC, SSC, threshold and PMTs voltage.
11)Select compensation controls and click “Run wells”.
12)Select the Specimen well and click “RUN Plate”, or select the wells you want to run and click
“RUN Well(s)”
13)At the end of the acquisition a box will appear that run is finished 14) To clean go to HTS menu > clean > cleaning plate
15)Fill the cleaning plate with:
a. Wells A1 – A4 with ≥ 250 µL fresh Clean Solution b. Wells B1 – B4 with ≥ 250 µL MilliQ H2O
16) When finished clean the plate with water.
17) Loose the HTS sample coupler from the instrument and place a tube of water on the SIP.
18)Leave the instrument in STANDBY mode.
