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?

AF647 is excited well by the red ~633nm laser and emits at a slightly longer wavelength. FAM (essentially FITC) is excited well by the 488nm laser and again emits with a slightly longer wavelength that can be detected with the FITC detector on the CantoII. There is no need to split these.

20190326 - Do you know when the next billing cycle is for the FlowJo site license option at Westmead Cytometry?

The FlowJo site licence billing cycle starts on the 1^st of March.

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?

The 2 colour samples in this scenario could be called FMO controls (fluorescence minus one controls). There can be a use for these in determining gating positions, depending on the amount of fluorochrome spilling over into other channels. If the spillover is high then yes FMOs would be useful.

20190122 - Do we need sodium azide free ABs for cell sorting?

Most pre-conjugated antibodies contain sodium azide to minimise bacterial contamination. This is an excellent quest ion and the answer is dependent completely on the researchers requirements.

Close to all current Westmead Cytometry researchers who are sorting their cells for downstream functional assays do not specifically stain with azide free antibodies. The current idea is that the concentration (once cells are stained) is low, followed by all the washing to remove unbound antibody that their is minimal azide that remains with the cells.

You can purchase endotoxin tested and azide free antibodies (eg. NA/LE format) but commonly for research cell sorting this isn't common.

20190129 - Can I transfer my FlowJo license to a new computer?

Yes you can. All we need is the hardware address of your new computer.

20181105 - Where did the cytometer configurations go?

We are currently renewing baselines and will have the printouts up soon by the instruments. The online versions are update however in the interim.

Cytometer Configurations

20181009 - What comp beads can I use for the Annexin/PI kit for apoptosis detection.

For PI/AnnexinV kit, there are no beads that bind PI, so actually using cells would provide adequate compensation for both AnnexinV and PI. Trying AnnexinV on colours other than the commonly used FITC (APC, BV421), as well as using DAPI instead of PI (not if using AnnexinV BV421) can also provide better resolved populations.

20180913 - Can I get autonomous (after hours access - still subject to building access - on the flow cytometers)?

Yes. If you have accumulated 10hrs or more of experience (used time) on the instrument we can instantly grant you autonomous access.

If you haven't, follow this link (https://goo.gl/forms/jBYZPG5bWf3bnYxj2) , complete the quiz, and email wrh.flow@sydney.edu.au for access.

20180724 - Why can I not calculate compensation on DIVA?

The software looks for a P1 gate on a bivariate FSC and SSC plot. It then also requires a P2 population for positive and (if not using a universal negative) a P3 population for negative events for each fluorochrome. A universal negative will only use events that fall under the P1 gate.

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).

Import your compensation controls.
Calculate a compensation matrix as per usual.
Click 'Calculate SSM'.
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

*update - 20180704 - 'B530_30B' PMT replaced. Instrument aligned. All specifications exceeded. New baseline recorded on the 4/7/2018 called WMI_2018_3B_6V_3U_3R_4Y.

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

If making a new experiment, proceed as normal.
If duplicating an existing experiment from prior to 4/7/2018, click continue when the configuration mismatch warning appears - you may wish to make a new experiment at some point as this will have the new configuration as default.

If continuing application settings see below (otherwise make a new experiment and proceed as normal).

The current suggested workflow involves

Seek assistance from WRHFlow staff if not 100% sure of the following procedure (some users may not have access to switch configurations).
Switch to the instrument configuration with the baseline recorded on the 29/11/2017 labelled 'WMI_2015_3B_6V_3U_3R_4Y' from the current baseline recorded on the '04/07/2018' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
Run performance check 'CST' using bead lot #70466 on 'WMI_2015_3B_6V_3U_3R_4Y' configuration
Once completed, there should be an error detected with the UV laser. Click OK and close the CST window.
Create a new experiment.
Apply application settings. Note the application settings applied should have been created between 29/11/2017 and '04/07/2018'.
Record all voltages using a screen shot.
Switch to the instrument configuration with the baseline recorded on the '04/07/2018' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
Create a new experiment, enter the voltages from the screen capture into the cytometer settings.
Save these application settings as updated application settings that will now be linked with the baseline recorded on '04/07/2018' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
Proceed as normal applying the updated application settings to your experiments.

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

Seek assistance from WRHFlow staff if not 100% sure of the following procedure.
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'
Run performance check 'CST' using bead lot #70466
Once completed, there should be an error detected with the UV laser. Click OK and close the CST window.
Create a new experiment.
Apply application settings. Note the application settings applied should have been created between 29/11/2017 and 'TBA date'.
Record all voltages using a screen shot.
Switch to the instrument configuration with the baseline recorded on the 'TBA date' labelled 'WMI_2018_3B_6V_3U_3R_4Y'
Create a new experiment, enter the voltages from the screen capture into the cytometer settings.
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'
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

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

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.

FlowJo is installed on the analysis PC that is bookable through PPMS. It is known as 'Workstation_Flow Data Analysis PC '
We can provide 'remote' access to FlowJo through Argus - email flow for more information - currently only available to site licence users.
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.

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