Flow Cytometry User Group Meeting – 8th August 10am – Conference Room C2.22
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We would like to revive the Monthly Cytometry User Group meetings as a forum where users can discuss cytometry related problems, issues, triumphs and successes.
This will be an open forum where users are encouraged to bring data along to share and work through.
The first one will include “Cytometry Tips & Tricks” as well as some discussion in regards to building/sharing core panels to be utilised across labs.
When: August 8th 10am-12pm.
Where: Level 2 Conference Room, WIMR.
What: Cytometry Tips & user discussion.
FlowJo Seminar Recording @ WIMR 15th July 2019
Link to download is here - https://cloudstor.aarnet.edu.au/plus/s/PrAnmHbblUS3vVV
FlowJo Seminars: Tools for Data Analysis
Date: Monday 15th July, 2019
Location: Room C2.20 and C2.22
9:00am – 10:30am Beginners | Introduction to FlowJo
10:30am – 11:30am Intermediate | Dimensionality Reduction and Clustering Plugins for Discovery
11:30am – 12:00pm Introduction to scRNAseq Analysis in SeqGeq
Sessions presented by: Tim Crawford, Field Application Scientist, FlowJo
Did you say colourful? BD Symphony is outputting high quality data.
The Symphony is ready to use! The instrument has been used by a number of researchers with positive feedback. The current configuration allows for at least 34 (yes 34) different fluorescent dyes to be detected in addition to 3 scatter parameters. Be sure to check out the current configuration listing the non-exhaustive plethora of dye choices.
Why would you want to use it? Well, if you are having trouble choosing fluorochromes for your panel, the increased options certainly may help, or if you are finding it difficult to find time on the Fortessa/LSRII, all currently utilised flurochromes can now be detected on the Symphony ensuring an event-free (*no pun intended*) transition. The additional scatter parameter can help also increase dynamic range allowing both small and large particles to be detected simultaneously.
Training takes ~30minutes if you already cytometer access and requests are accepted through PPMS.
Spectral Unmixing - Improve your population resolution…
FlowJo 10.6 was released yesterday (after months of beta testing)! This update allowed weighted non-negative least squares fitting for spectral unmixing of your cytometry data!
What does this mean… Instead of compensating your data, you can now spectrally unmix, essentially using all the detectors as reference points for the fluorochromes spectra.
You will need too (and should have been already) ensure all instrument detectors are active and collecting signals as well as ensuring all signals are not off scale for non-primary detectors.
The process is almost similar to compensating using FlowJo but just involves clicking 2 more items in the software when compensating your data.
And you guessed it – this is where the Symphony excels with its numerous detectors.
The shared data analysis computer has had the upgrade but see here for further information.
Additionally this can be performed retrospectivly on standard cytometers if the data was collected with additonal detectors enabled.
Example spectral data - BV737 detected across many detectors.
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Image Cytometry
‘delightfully detailed dots’
I recently had the opportunity to acquire samples using an image cytometer for a research project using an ‘Imagestream’, accessed via Sydney Cytometry at the CPC. An image cytometer will generate images for the events that are collected. The instrument I used outputs not only multiple fluorescent high quality images that can be matched to specific dots in traditional flow cytometry data, but provides automated image analysis modalities for data mining. Despite the release of initial variants of the technology way back in 2004, it is an often overlooked and underutilised tool in the cytometry toolkit. The spatial localisation of fluorescence signals is incredibly powerful in the addition of features (similar to parameters in traditional flow cytometry) for sub setting populations. If you are interested in image cytometry please contact westmead.cytometry@sydney.edu.au to understand how this technology may add to your research and how you can access the technology.
Levitas LeviCell Trial – ITS HERE NOW – February 22nd to March 5th 2021
‘live cell separation using microfluidics, magnets and MRI technology’
Westmead Cytometry will host the LeviCell for approximately 2 weeks for interested researchers to trial the technology. The small instrument (0.7mx0.3m) essentially allows for the rapid bulk isolation of live cells from debris and dead cells. There has been considerable interest for the potential of this instrument to slot into workflows where cell dissociation from tissue samples are a necessity. An evaluation of the system will follow in the next newsletter but if you would like to evaluate the system please email your interest as there is availability but reagent limitations.
Unused Analyser Session Charges
Starting on the 1st of March 2021 (to be delayed and now with 3 strike rule in place), booked but unused sessions on the analysers will be charged at 25% of the hourly rate as per the published 2021 Westmead Research Hub user fee schedule to discourage users reserving instruments during peak periods with usage not occurring. The charge is primarily for users who unfortunately book but do not show up resulting in a disadvantage to all users.
Westmead Cytometry User Group Meetings
‘2nd Friday of the month @ 11am – click to add to your calendar!’
A forum to communicate with fellow cytometrists in an informal, friendly and ultimately thought provoking environment. The user group meetings will consist of a small delve into a research project/topic followed by an open session with the aim to improve and discuss researchers concerns right from sample preparation to data analysis. The schedule for 2021 is included below and will be held in the level 2 WIMR seminar rooms.
Fluorochrome Degradation & Compensation Options
‘validation is key’
I thought it useful to outline (at least on the instruments at Westmead Cytometry) different approaches users are implementing when deciding on the practicality of generating for high parameter compensation controls.
Our recommendation is to generate compensation controls with fluorochromes that have undergone the same process as your sample (and in parallel), followed by validation that the spectral profiles of the compensation controls does indeed match the spectral profiles of the flurochromes used on samples acquired (by ensuring populations are not over/under compensated, along with identification of intended populations).
However with the increase in high parameter cytometry popularity and practical/financial constraints there have been variations to the above recommendations observed. These variations include
-fixing compensation controls with acquisition on subsequent days
-unfixed compensation controls with acquisition on subsequent days
-utilising a compensation matrix across different days with no application settings applied (and 8 peak instrument checking)
-utilising a compensation matrix across different days with application settings applied
-acquiring samples on days after staining with compensation controls made on the day of sample acquisition
etc.
It is important that in the above listed variations that the compensation matrix applied to the samples is validated not to change populations identified. The good news is that the validation is quite straightforward and generally requires the acquisition of the same compensation controls at different timepoints. In addition, a change in the matrix does not necessarily mean the entire set needs to be remade. There have been cases where antibody conjugated fluorochromes were stable but viability markers were not, and other examples where the switch of certain fluorochromes from beads to cells resolved the changes observed on subsequent days.
The take home message is variations to the recommended workflow are occurring and as long as validated to not alter the matrix applied to the sample causing population changes then they are valid approaches.
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Fancy Using A Supercomputer? I did (and loved it).
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