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Implementation of Application Settings

1. Import the Previous Experiment into the Diva Database which was run for the same cell type/preparation with the Application Settings

2. Open the Previous Experiment. Duplicate it without data

3. Rename a New Experiment with today’s date

4. Transfer the Cytometer Settings from the “CST Adjusted” tube of the Previous Experiment (Open the Previous Experiment. Copy the Cytometer Settings from the “CST Adjusted” tube) to the Global Cytometer Settings of the New Experiment (Open the New Experiment. Paste the Cytometer Settings into the Global Cytometer Settings of the New Experiment)

5. Define the changes of the PMTs to keep the same CST target values between the consequent experiments:
a. Take the tube of CST beads from the DWB211 fridge (if not there – ask FCRC staff for the CST beads)
b. Record data in the CST Old tube
c. Go to the next tube (CST Adjusted):
i. Start the acquisition of the CST beads and adjust the PMT voltages until all the histograms fit into their gates
ii. Record the data
Note 1: DO NOT modify the Interval Gates on the histograms
     Note 2: Only modify the PMT voltages if necessary
     Note 3: If the difference in the PMT voltages for any of the parameters exceeds 10 volts, please make sure to notify the FCRC Staff in order to make corrections if needed

6. In order to apply the adjusted Cytometer Settings to the entire experiment:
a. Copy the Cytometer Settings of the CST Adjusted tube
b. Paste them into the Global Cytometer Settings

7. Record all compensation controls. Calculate the compensation, “Link & Save”
Note 1:  From now on DO NOT modify the PMT voltages of the fluorescent detectors
    Note 2: Make sure to modify FSC and SSC PMT voltages (only) if needed to accommodate for the scatter of the cells or compensation beads

8. Record all experimental tubes
Note: Make sure to modify FSC and SSC PMT voltages (only) if needed to accommodate for the scatter of the cells

9. Once finished with the acquisition of all the experimental tubes proceed to
Data Management according to the explanation and scheme at http://www.rockefeller.edu/fcrc/data/BDLSR and Instrument  Maintenance at http://www.rockefeller.edu/fcrc/tips/BDLSRII-maintenance