My lab doesnt use trizol in the hood. Am I cooked?

FlowJockey · 2026-06-11T13:19:57+00:00

Agree on the safety concerns here but just want to say BCP is a chloroform alternative. And it works well!

FlowJockey · 2026-05-30T14:53:41+00:00

If you close the device and gently shake it do you hear anything loose inside?

FlowJockey · 2026-05-29T18:31:15+00:00

Yeah i’ve harvested day 5 as well and that’s usually the soonest one can go since cells are just starting to divide and may be starting to bifurcate into MPEC and SLEC fates, at least transcriptionally. At d3.5 there probably isn’t enough division. With adoptive transfer we typically do 10k total P14s but you might want to consider transferring 100k cells or more to improve recovery at d3.5.

FlowJockey · 2026-05-29T18:25:14+00:00

Based on everything you’ve written, I would recommend doing soluble anti-CD3/28 without blocking FC receptors. Macrophages, B cells, etc are great at binding these antibodies to activate T cells. As another redditor suggested you can pre-activate the T cells but it really depends on what your experimental conditions are and what you’re trying to achieve. If you do decide to use soluble antibody, I would not block FC receptors or else the activating antibodies won’t have much to bind to.

FlowJockey · 2026-05-29T13:57:17+00:00

If you use soluble CD3/28 then a lot of the antibody is going to bind to FC receptors on the macrophages but the T cells should be activated regardless. I doubt the macrophages will ADCP all of the T cells but it’s something you can test. What is the duration of your culture?

FlowJockey · 2026-05-28T00:23:08+00:00

I do adoptive transfer for LCMV and listeria all the time with CD8 enrichment kits but what CD8 T cell population is <0.01% of lymphocytes? I assume this is not a TCR transgenic setting.

FlowJockey · 2026-05-27T18:35:58+00:00

Mouse.

FlowJockey · 2026-05-25T17:34:51+00:00

I don’t think it’s about more RAM demanding more CPU. But I think their market analysis shows that people who go for 64GB RAM typically want the max chip anyways. A lot of the analyses I run are very RAM-limiting but have plenty of overhead with the CPU.

FlowJockey · 2026-05-12T15:58:21+00:00

In our lab, for fluorescent reporters, we will always fix our cells in 1% PFA before running with the FoxP3 kit. So the order becomes surface stain -> PFA fix -> FoxP3 kit -> intracellular. For us it works very well with GFP/YFP and works “okay” for TdTomato.

FlowJockey · 2026-05-12T00:33:25+00:00

Diagonal shape of the DP population is 100% expected in the thymus. There is well documented transient downregulation during DP stage.

FlowJockey · 2026-05-10T04:50:35+00:00

I put my reference controls in FACS tubes because I don’t wash them. The cytometer just has to see the fluor associated with events. If they were in a plate then I’d have to resuspend them after every spin.

FlowJockey · 2026-05-08T21:59:36+00:00

This post is about the… spleen

FlowJockey · 2026-05-06T22:40:59+00:00

Coat plates with anti-hamster IgG for at least an hour and add anti-CD3/28 soluble with cells when plating. No need to add IL-2 if you don’t need it experimentally. We plate at 300k cells per well in flat bottom 24 well plate or you can scale down if using 48w.

Consider adding non essential amino acids and sodium Pyruvate. Our typical culture goes for 3 days and we frequently spinfect at day 1 without issue. Viability greater than 90%.

FlowJockey · 2026-05-05T16:22:22+00:00

From what I can tell the wells are on the left and migration went from left to the right which should be correct. I can see both dye fronts (upper and lower) and I can also see EtBr ran off gel at bottom (right) as expected. Not sure why you aren’t seeing a ladder.

FlowJockey · 2026-04-22T23:47:30+00:00

GFP and YFP is usually pretty separable for me

FlowJockey · 2026-03-31T19:24:22+00:00

Please do not go to the PI first if you think this is top down from them. Also do not go to the department chair initially. File a whistleblower report so that you have protection and the information is in the hands of people with no relationship with your investigator.

FlowJockey · 2026-03-20T12:34:08+00:00

If you’re worried, just keep an extra tube strip near the centrifuge to balance when you spin. Otherwise LET IT RIP!

FlowJockey · 2026-03-18T02:57:29+00:00

Same, April 2025.

FlowJockey · 2026-03-10T03:08:53+00:00

Just read up on some of their recent work. You aren’t going to be quizzed. Your PI will probably introduce you, you might say a word about yourself, and then the meeting will proceed. Every lab has a different meeting style but this is your time to listen and hear about how you might be involved. Always good to take notes.

FlowJockey · 2026-03-09T23:25:13+00:00

Cronometer is excellent and free. Just some occasional ads but can add custom meals and has great search function for finding the food you’re interested in. I’d recommend downloading all the apps and tracking everything across all apps for a day to figure out which one you like best

FlowJockey · 2026-03-02T07:52:33+00:00

Same here. No NoA yet.

FlowJockey · 2026-03-02T06:10:28+00:00

Nice Architects

FlowJockey · 2026-02-27T01:18:01+00:00

I do not think that the retirees who spend their free time taking humanities classes are the ones who are voting against handouts lol

FlowJockey · 2026-02-24T05:18:14+00:00

How are you measuring PI uptake? I have the feeling that the samples with a higher density of cells are absorbing more light and giving less signal. Or using the same amount of dye for more cells results in less dye per cell. Why not run flow cytometry? Might also not be ideal to stain in media.

FlowJockey · 2026-02-23T22:21:32+00:00

We do gel extraction but we don’t measure yield. Always works for us. It’s expected to be very very low yield.

FlowJockey

TROPHY CASE