20180621 - What software can I use to calculate a spillover spreading matrix?

WRHFlow recommends calculating the spillover spreading matrix using a recent version of FlowJo - v10 (FlowJo v9 for MAC also has the functionality).

1. Import your compensation controls.
2. Calculate a compensation matrix as per usual.
3. Click 'Calculate SSM'.
   
4. Click 'SSM' and then 'Display or Export SSM'. Note the difference between the compensation matrix and the spillover spreading matrix.
   

20180612 - How much notice do I need before cancelling a sort.

We prefer the most notice possible when cancelling a sort. We will only charge a sort if we have started to setup the instrument (this typically means a cancellation with 24 hours notice will not get charged and a cancellation in the morning of the sort day, if we get the email in time will result in no charge). We also don't charge if we have setup the sorter but can use it for another sort. We will charge for a cancelled sort if the notice was late and the instrument is not otherwise used. 

20180612 - LSRII UV laser 

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.

*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. 

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

Yes. We have mapped the network drives now using a service account 'svc-public'.

All you need to do is:

• Open 'Finder'
• Click 'Go' and then 'Connect to server'
• 
• Connect the required network drives. 
• 

20180530 - The UV laser was replaced on the Fortessa. What do I need to know?

The UV laser has recently been exhibiting higher than liked CVs. We have recommended avoiding use of the UV laser on the Fortessa for the last week due to CVs that were above 10% (Instrument specification is below 6% - we aim for <3%). A new UV laser has been installed which resulted in CVs of <2%, and improved power from this laser line. All lasers were additionally aligned and are performing well.  

Application settings...

For those users using application settings, we have modified the baseline expiration date to allow continuation of the current application settings in use.

In regards to the UV laser change, please note that you will need to review voltages for the parameters off the UV laser line. 

The current suggested workflow involves

1. Applying previous application settings as per usual and reviewing (not updating) UV signals. Please note we do not expect you to have to change any voltages. Please ensure you review the signals with compensation turned off as this was causing some confusion as signals looked like they had changed when they really hadn't. 

This is suggested as other parameters should have been adequately updated when your previous application settings had been applied.  

We recommend that you make note of the UV laser change in your experiment.

We have currently decided not to redefine the baseline to minimise impact to users (and other reasons including the baseline being validated on a different configuration).

We also strongly recommend recording new compensation controls.

20180530 - Is there any way for me to gain access to FlowJo?

Yes. There are 3 ways we enable access to FlowJo. 

1. FlowJo is installed on the analysis PC that is bookable through PPMS. It is known as 'Workstation_Flow Data Analysis PC '
2. We can provide 'remote' access to FlowJo through Argus - email me for more information - page to be updated soon. 
3. We can issue FlowJo licences through the FlowJo Licencing Server. These must be associated with a valid project. Current charges can be found on the charges link to the left. This means that you can use FlowJo on your local computer although this is a charged option.

20180529 - What criteria do I need to meet in order to get after hours booking access to the cytometers?

An email sent to flow requesting autonomous access. Access is granted if >10hrs usage. Alternatively a quiz pass result where we ask you some questions to ensure you understand basic operation. 

20180523 - How to acknowledge WRHFlow

Question: Would you please let me know how we should cite the flow cytometry facility for publication/thesis?

Answer: Flow cytometry was performed in the Flow Cytometry Core Facility that is supported by Westmead Institute, Westmead Research Hub, Cancer Institute New South Wales and National Health and Medical Research Council.

20180522 - Bringing cells to be sorted

When we need to bring you samples how do we bring them? See here. 

In an eppendorf tube? In media? In PBS? - answered in link above. 

How much volume? See here.

On ice? Recommended - generally though cell type specific.

What else do you need us to bring? Collection tubes with something to collect samples into - see above link. 

 And whereabouts are you actually located? Great question - if you don't have access to WRHFlow - ask @ WIMR reception and they will contact us and we will escort you to the sorter. If you do have access we should generally be around the labs or the office - L2.02, L2.04,L2.05,L2.06.

20180521 - What does signal below zero on a fluorescent parameter mean and should I gate only for events > 0 fluorescence?

Do not only gate for events greater than zero. Values less than zero are technically correct on digital instruments. See this previous post about negative fluorescence. Also note acquisition inconsistencies during acquisition indicating possible sample issues during acquisition.

20180521 - Cleaning up data

Problem:Is there a recommended order for cleaning up data - e.g. time, single cells, live/dead, lymphocytes to get he best data quality output?

Outcome: All orders will result in the same populations being displayed (the final product will be identical despite the order of the gates). 

20180518 - Where can I find instrument configurations?

Instrument configurations (and very soon), spillover spreading matrix information can be found on our site - Cytometer Configurations.

20180517 - Where do I enter my FlowJo licence number?

Here...

20180517 - Negative amine reactive bead compensation issue

Some users have reported not seeing 2 distinct populations when compensating using amine reactive beads. If this happens 1 easy fix is to aliquot some negative beads into a separate

20180517 - Negative amine reactive bead compensation issue

Some users have reported not seeing 2 distinct populations when compensating using amine reactive beads. If this happens 1 easy fix is to aliquot some negative beads into a separate tube, and acquire these appending the data file. This way there should be a definite negative population and a positive population that you can gate for compensation. 

20180517 - GFP & PECY5 detection on the CantoII

Yes these two markers can be both identified and distinguished on the CantoII.

20180517 - Compensation control tips

Compensation controls are recommended for any panel using more than 1 fluorescent marker.

Things to remember - 

• The software requires a positive and a negative sample, with the only difference being the fluorescent molecule you are compensating for.
• A universal negative can only be used (both in DIVA and in FlowJo) if all compensation controls use the same type of sample. For example all comp beads from company X, all cells, etc. 
• A universal negative can not be used if there is a mix of particles, e.g. comp beads + amine reactive beads, comp beads company X + comp beads company Y, cells + beads.
• Ensure you select the entire negative population if you are drawing a P3 gate when not using a universal negative setup in FACSDiva (see WIMR-SWP-OP-WS-504.01 Flow Cytometer Analysers.pdf). To do this ensure bi-exponential display is activated.
• Your compensation control positive median signal should be brighter than the brightest cell expressing that colour. 

Any questions please contact WRHFlow!

20180517 - User access hours

User access hours are as follows (confirmed April 2018) - WRHFlow Access Hours.pdf

If your card doesn't work as stated please email wrh.flow@sydney.edu.au

20180516 - DAPI / BV421 use on the sorters

On the ARIAIII BV421 is detected using the V440/40 detector making DAPI incompatible. On the Influx BV421 can be detected off the 440/40 with DAPI being better excited off the UV with detection using a 460/50 filter off the UV laser line. It is good practice to gate while looking at both channels to avoid dumping any BV421 positive events that may be misclassified as dead cells. However this is normally not too much of a problem. See Cytometer Configurations for instrument configurations. 

20180515 - Fluorescent protein

Problem: User is already using mCherry & GFP. User requires a third colour, but wants to leave AF647 empty and BFP has been tried and it too dim. 

Outcome: Recommended if not on the same cell, dTomato for the current cytometer configuration. If it isn’t urgent in June we should have a 445nm & 594nm laser, and this would add eCFP & mRaspberry. Useful link for not so common fluorophores - http://learning.clontech.com/spectra.html

20180514 - Inflammatory samples

Problem: Today we had a user who was analysing data from ileum samples who had trouble identifying CD45+ live myeloid cells.

1000000 events In both the in-flamed and un-inflamed samples had been acquired. In the inflamed a easily identifiable population of CD45+ lymphocytes could be identified however in all the non-inflamed tissue no equivalent population could be observed. 

Outcome: Inflamed samples contain a much higher cell:debri ratio it was easy to see the population with a 1000000 events. In the non-inflamed the ratio of cell:debri was much lower. Luckily to prove this, one can concatenate all replicates of non-inflamed, and use this concatenated data file to observe similar lymphocytes populations putting our minds at ease that the live CD45+ actually existed in the non-inflamed tissue albeit at a much reduced frequency. The lesson is to consider how many targets cells you are recording and not only the total events, especially in samples that can be highly variable in cell:debri ratio.