20190517 - I am planning to do Flow-FISH using 3-colour system. I have LDS-751 and EdU-click conjugation with AF647 and PNA-probe conjugated to FAM. I’m wondering if it might be possible to do this on the CantoII? Or do I have to split my colours up into two experiments?

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The FlowJo site licence billing cycle starts on the 1^st of September and the quarters are as follows.

Sep-Nov

Dec-Feb

Mar-May

Jun-AugMarch.

If licence is ordered at a later date it'll be charged pro-rata by month.

20190304 - We are planning a three colour FACS Canto experiment. I plan to have a no stain, and single stain controls. Should I also have 2 colour controls?

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We strongly recommend that you make note of the UV laser change and the B530_30B PMT change in your experiment records, review the UV parameters and other parameters and record new compensation controls.

*update - 20180629 - R780_60A detector fixed.

*update - 20180628 - The old LSRII UV laser had been exhibiting a declining UV laser power output. This occurs over time as the laser was >4 years old. After repeated presentation of performance issues, and a measured output of 9.8mW (specification is 20mW), the engineer replaced the UV laser on the 28th of June 2018. The new UV laser is exhibiting excellent performance (19.8mW output and a CV below 3% on the primary detector) but we now need to confront the reality that the baseline has changed and how this affects current application setting users.

Currently 2 other detectors (Blue - B & Red - A) are not performing perfectly (but are still very usable) and hence we have not recorded a new baseline. We will attempt to solve this ASAP. Currently we are continuing with the existing baseline recorded on the 29th of November 2017 because of this. The QC correctly identifies that CST has failed and that the UV detector voltages need to be reduced by ~100 volts (due to the new laser installation). This means that if you are using application settings, the instrument will set your detector voltages ~100 volts lower in order to put the positive peak in the same spot as what it was. This will result in your negative population being lower and/or less spread for the current application setting users. It is recommended that new compensation beads be recorded for any experiment. 

We do plan on recording a new baseline as soon as the instrument is performing perfectly. We will notify users, as well as update the site/configurations once this occurs. Once this has occurred, researchers not currently using application settings will not be affected and can enjoy better signals off the UV laser. Users who are currently using application settings and who wish to continue them, the below option is the recommended approach. 

It is recommended to note the UV laser change date for your reference.

Continuing Application settings (this only will apply once the above 2 detectors have been fixed and a new baseline recorded with subsequent notification via the information screen that displays after logging in to the analysis PC)...

The current suggested workflow involves

1. Seek assistance from WRHFlow staff if not 100% sure of the following procedure.
2. Switch to the instrument configuration with the baseline recorded on the 29/11/2017 labelled 'WMI_2015_3B_6V_3U_3R_4Y' from the baseline recorded on the 'TBA date' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
3. Run performance check 'CST' using bead lot #70466
4. Once completed, there should be an error detected with the UV laser. Click OK and close the CST window. 
5. Create a new experiment.
6. Apply application settings. Note the application settings applied should have been created between 29/11/2017 and 'TBA date'.
7. Record all voltages using a screen shot. 
8. Switch to the instrument configuration with the baseline recorded on the 'TBA date' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
9. Create a new experiment, enter the voltages from the screen capture into the cytometer settings.
10. Save these application settings as updated application settings that will now be linked with the baseline recorded on 'TBA date' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
11. Proceed as normal applying the updated application settings to your experiments.

We strongly recommend that you make note of the UV laser change in your experiment, review the UV parameters and record new compensation controls.

*update - 20180613 - The UV laser was able to be aligned to bring signals levels back to within acceptable specifications. Plans have not been made to replace the UV laser. 

*original post - 20180612 - The UV laser has recently been exhibiting a declining UV laser power output. This occurs over time as the laser is now >4 years old. Currently it is (and we) still recommend the current use of the UV laser on the LSRII, however we have scheduled for an inspection for the 13th June 2018 that may result in a UV laser change. If this occurs we will update users and provide our recommended procedure for users currently utilising application settings. Updates will be posted here.

20180612 - Can I access the 'Public' & 'Scientific Platforms' drive on the MAC analysis computer in C.2.34

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