Steps for Acquiring Data

Typical workflow for setting up a BD flow cytometer (with compensation)

Start up the system

1. Start up the cytometer and computer.

2. Start BD FACSDiva software

3. Check fluid levels in the Cytometer window and prepare tanks as needed

4. Verify that the detectors and optical filters are appropriate for your experiment

5. Check that laser warmup has finished.

Create an experiment in the Browser.

1. Create Browser elements.

2. Create the plots, gates, and statistics needed for recording. Use the full log scale (i.e the log 5) and remember biexponential scaling is very helpful and helps with seeing the true effect of compensation.

3. In general, your parameters:

  • FSC and SSC: linear; select A (Area), H (Height) and W (Width) for doublet discrimination.
  • Fluorescence will generally be acquire in log
  • ***An important exception- if doing cell cycle analysis, this fluorescence must be acquired on a linear scale!

Generate the population hierarchy, and gating strategies.

4. Enter information in the Experiment Layout as needed. Under experimental layout you can add labels for your markers, generate tube specific workspaces and acquisition numbers.

5. Apply application settings to the cytometer settings or set parameter voltages. Application settings allow standardization of PMT voltages across instruments and days and is very useful for longitudinal studies.

Set up Compensation

Compensation is an essential part of any multi-color experiment. In order to properly compensate you will need an unstained control and a single stained control for every color in your experiment.

1. Select Experiment > Compensation Setup > Create Compensation Controls.

2. Create label specific controls if necessary

  • Click Add
  • Select fluorophore from the list

3. Install and run the unstained control tube.

4. Install and run the stained control tubes.

5. Record and view the data, and gate the positive populations.

6. Select Experiment > Compensation Setup > Calculate Compensation.

7. Rename the compensation setup and apply it to the experiment.

Record experimental data

1. Install tube and select “Acquire Data”.

2. Verify proper running of sample and select “Record Data”.

3. Repeat for each sample.

4. Export the data as FCS 3.0 or 3.1 (not 2.0) files and the experiment.

5. Run a batch analysis to generate the pdf file of your samples and export the .csv file of your statistics.

6. Export the experiment as an experiment template.

7. Delete the data from the computer so ask not to lose your data if the computer crashes and also to prevent problems with Diva.

8. Clean the machine! Run Coulter Clenz for 2 minutes, 10% bleach for 2 minutes, and MQ water for 5 minutes.

9. Shut down the system if you are the last user at 4pm, regardless if someone else is after you.

 

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