Doing more with less: Improving flow cytometry staining

Our latest paper isn't going to cure cancer. But it may just making research into curing cancer 10-fold cheaper. A major cost for immunology, oncology and haematology labs is antibodies, especially with the fancy (and expensive!) new dyes coming on the market, which let us hit the 40-50 parameter range in flow cytometry. Fortunately, Dr Oliver Burton in our lab has developed a simple and easy approach to reduce those costs by 10-fold, while also improving the quality of the data: shifting to overnight staining. You can read the full paper here, describing the protocol, fixatives and optimisation approaches, but essentially, lower antibody concentrations give less background, and if left overnight to stain are able to give even better signal detection. It is also a game-changer for wet-lab staff: rather than having all day dissections and staining, and then cueing for flow cytometer hours in the evening, you can set everything up, put on the staining and get an early night. Next morning your cells are ready to go! 

Out now at Current Protocols!

Update on Thursday, November 17, 2022 at 4:54PM by Adrian Liston

We have had multiple requests for a detailed staining protocol, so here it is!

Congratulations to Dr Ana Acosta!

Well done to Dr Ana Acosta, who successfully defended her PhD today! Ana tackled a challenging and exciting project on the role of HNF1A in monogenic diabetes, generating a new mouse model and validating results in primary human islets. Her work dramatically alters the way we see HNF1A in glucose homeostasis and diabetes. A very productive PhD, performed at University of Lille, with Prof Caroline Bonner, and the University of Leuven, with my team. The 20th PhD student to graduate from my lab, and one of the last from our Leuven days! Great job Dr Acosta!

New cause for primary immunodeficiency discovered

Our lab has a new study on primary immunodeficiencies out now at Cellular & Molecular Biology! We studied two families with combined immunodeficiency and found mutations in the Calcium channel ITPR3. The mutations reduce the function of the channel, making the channels 100-fold less capable of initiating a Calcium flux after cellular stimulation. T cells from the patient had poor responses throughout the signalling cascade: reduced Calcium flux, poor nuclear localisation of NFAT1 and reduced proliferative burst, explaining the impeded response to infections. The most severe patient required a bone-marrow transplantation to correct the defect, while the other patient is doing well with regular IgIV treatment. The work established ITPR3 as a new cause of primary immunodeficiency, after previously assuming that these Calcium channels had too much redundancy to be a cause of genetic disease. Read the full paper here, or take a look at the illustrated abstract below for a short-cut summary!

Using gene delivery to protect against diabetes

Exciting new paper out from the lab on using gene delivery to protect against diabetes. The work is based on the "fragile beta cell" hypothesis, which postulates that some individuals are prone to diabetes because their beta cells are more prone to fail during stress situations. We previously demonstrated that the Glis3-Manf axis was central to dictacting how robust or fragile beta cells were, during stresses either immunological (type 1 diabetes) or metabolic (type 2 diabetes) in origin. Based on this data, we designed a gene delivery system, which essentially tricks beta cells into making more Manf and becomes robust in the face of stress. NOD mice, treated with this gene delivery of Manf, become resistant to diabetes. As the gene delivery system we use harnesses the endogenous insulin promoter (specific to beta cells, and upregulated during cellular stress), we can use low doses of the gene delivery system delivered intravenously, without altering the rest of the body. This gives the system a high potential for clinical translation. Read the full paper here, or check out our illustrated abstract below.

Using "I language": how to communicate with your team

One of the biggest challenges of having your own team as a PI is learning how to communicate with them about behaviour you are unhappy with. For me, this is often about deadlines. I hate deadlines being missed, and in particular I hate it when a deadline is missed and not communicatd, and basically it is on me to realise, chase up with the person, diagnose the problem and arrange a new deadline. Because it annoys me so much, I go out of my way to make the deadlines achievable - I ask my team member how long they think a task will take, and then I'll often add on a few days or weeks if I think they are under-estimating it (or if the deadline would fall during a period where I wouldn't be able to respond anyway). I'm also highly receptive to a team member telling me, in advance, "I know that we agreed on X as a deadline, but I can already tell I won't make that, can we push the deadline back to Y". With all that being the case, some people repeatedly miss deadlines, and it really grinds my geers. It is one of the easiest ways to sour a professional relationship with me (which, incidently, I explicitly write in my "lab policies" doc that I give to new lab members - it is not a secret!). If it is frustrating me, then I need to bring up the issue with my team member and deal with it. Here is my secret:Use "I language". To explain "I language", it is easiest to start with "you language". "You language" is saying "you missed this deadline". The problem with this is that it immediately puts the person on the defensive: you'll see them coming up with excuses or rationalisations. That isn't a productive mindset for you to change someone's behaviour."I language" reframes the debate. "I feel really disrespected that you missed this deadline and gave the work to me late. I had reserved Friday morning to work on it, but instead I had to skip out on going to the park with my son on Saturday because I got this late. I know that this feels like a one-off to you, but when I manage a team of 15, people only need to do this twice a year to ruin almost every weekend for me. This is really important to me, and now that this has happened several times I feel like you don't respect me or value my time".Advantages of "I language":

• it is actually true. The problem that you are addressing isn't actually their action. The real problem is that this particular action hurts you. We all have different trigger points - messy bench, missed deadlines, swearing, having the radio on - each of these will cause one person to grind their teeth while another person literally won't notice. How do they know this is a sensitive issue for you without you telling them? 
• it emphasizes the importance of the issue. For some people, deadlines just aren't that important, so it is hard to make them feel like deadlines are important to them. But they can understand that missing weekend after weekend with your child is important to you, and once you link the two they will get it
• it puts the other person in a zone of empathy rather than in a zone of defence. They don't feel attacked, they feel taken into your confidence, and are gaining an insight into what is important to you
• word spreads faster. People don't like to share that they got told off, but they will share that you are really sensitive to some particular things
• it works. Except for sociopaths, people don't like causing pain to other people. If you find that "you language" works better on someone than "I language", that is a giant red flag that that person has no empathy or ability to work as a team

This probably sounds a lot more like relationship advice than mentoring advice. Which is because it is - I got it from "Why Marriages Succeed or Fail", a scientific analysis of relationships based on recording and analysing real interactions between couples and then correlating these interactions with later divorce rates. While the book is about personal relationships, I believe that the data holds just as true for professional relationships. It is well worth a read for other tips, such as making sure you have a good ratio between positive and negative feedback.

An international lab

Scientists have come from 45 countries across 6 continents to work together in our lab!

Lab BBQ

'Eureka moment' as impact of brain injury in mice reduced

Lab picnic

Brain Tregs on The Immunology Podcast

Listen to a great episode of The Immunology Podcast to hear a section on our recent paper about brain Tregs.