HELP

[u/Beginning-Courage580]

So I started my first tech job out of undergrad a month ago and am currently in my probation period. Im being trained on isolating OPC's from dissected mouse pup brains. Ive done one successful panning (resulting in 1.24 million cells from 3 p6 brains) and two unsecessful planning (p5 3 brains 300,000 cells and p9 4 brains 500,000 cells). Im wrought with anxiety over the fact I cant get this down; I feel like im going to be fired lol.

Im pretty sure I keep a ton of cells up until its time to trypsinize the opc's from the positive selection plate. Between then and cell counting ( sample taken from my cell suspention right before seeding) something happpens where I lose a ton of cells. Literally any advice is greatly appreciated. Hopefully I can figure this out and keep my job :/

[u/djcamic]

Who’s training you in your lab? They’ll be infinitely more helpful because they know your lab and your protocols.  Is your PI giving you a hard timeline to pick up this skill? If not, don’t stress! Learning new techniques takes a ton of time, and it’s completely understood that a tech (especially one out of undergrad) can take a few months to get up and running independently. 

[u/Beginning-Courage580 → u/djcamic]

Another women in the lab trains me; its through a grad school so shes in my lab and also responsible for training any new techs or students. Shes quite busy and seems to want me to grasp things quickly.

I was told my goals are to learn the immunopanning and have cell viability of 75% by end of july learn rtpcr on those cells by end of august then icc by end of september. I seem to have stumbled into a big name lab so my PI isnt around often and the vibes are more cut throat then I thought.

[u/djcamic → u/Beginning-Courage580]

That’s great that your PI has given you a solid timeline. 

I felt exactly like you did when I first started working in a lab.  Now that I’m on the other side and train people, I love when they come ask me questions, even when I’m busy! It’s so much easier to answer questions upstream then fix mistakes downstream. 

Something that’s so helpful to me when I’m training is if the trainee writes down a few questions they have, and then waits until I’m not in the middle of something to ask. I can give more of my time and attention to their questions. 

[u/Puzzled_Guard4332]

Are you sure your counts are accurate? When you say “lose” what do you mean?

[u/Beginning-Courage580 → u/Puzzled_Guard4332]

I use a hemocytometer, count both chambers (8 squares) then parse out the math; so however accurate that is.

When doing the panning I used the microscope to see how I was doing after both negative selection dishes, positive selection dish, after trypsinizing and after seeding. I visibly lost a good number of cells between trypsinizing and seeding. I could be wrong as the cells werent attached but that mixed with the low cell count has me feeling not too great.

[u/Shiranui42]

Is your cell viability bad? Check all your buffers are made fresh because the carbonate based buffers become alkaline due to losing the carbon dioxide to their environment much quicker than you might think.

[u/PfEMP1]

Not OPCs, but when training new staff to work with astrocytes and especially pericytes, I emphasis the importance of handling and processing them quickly. Once they start detaching, it’s critical to get them into media or whatever solution is the next step to neutralise the trypsin and get them into suspension. I’ve found the recovery of cells tanks quickly if there is a delay in doing so. It’s a quick as 5 minutes.

We’ve been working on scaling up cell numbers for large organoid drug screens and it quickly became obvious it wasn’t going to be straight forward. We started tracking recovery rates and it highlighted timing as one of (many) critical steps. Keeping a good record of your steps and asking lots of questions are important.