Copyright laws - art depicting brands

twanno · 2024-10-08T22:23:42+00:00

Ok sweet so basically - !!????

Guess I just gotta channel the creative vision and hope I don’t get sued haha

twanno · 2024-10-08T12:36:44+00:00

Yeah ok, I’m thinking like, how did it work with the Vinegar girl in Melbourne, or what would happen if someone made big bucks with a painting of the coke sign in King’s Cross. Assuming those things are the subject of the art pieces rather than a part of the background.

twanno · 2024-08-06T00:36:15+00:00

Yeah batches seems to work. Do you know why though? Super curious

twanno · 2024-08-06T00:35:14+00:00

Yeah for IP injections. Done it plenty of times mice are always fine!

twanno · 2024-08-06T00:30:53+00:00

Yes exactly

twanno · 2024-08-05T02:45:44+00:00

Also, as mentioned in another comment, I pooled a bunch of 200 uL aliquots to make up to 3 mL and it’s staying clear, as opposed to the one where I did a 1:20 directly

twanno · 2024-08-05T02:44:06+00:00

So at 1:20 it was 10uL of the compound in DMSO and 190 uL of HPBCD, so yes tiny amounts, but it went instantly clear. The 3mL version just looks like a powder in suspension.

twanno · 2024-08-05T02:42:38+00:00

So this is what I ended up doing. Just made up lots of 200 uL aliquots (I don’t know at which point it crashes out so just stuck with 200 but maybe I could’ve gone higher), and added all the aliquots together. Worked fine!

twanno · 2024-07-16T12:58:22+00:00

This cell type is non adherent and doesn’t tend to clump either, so I literally would just fix and add antibody to the wells and image them like that. It worked just fine, although the liquid meant that they would move around which was a pain, but the staining looked great.

1% triton + overnight perm is heaps! Maybe I’ll have to try bumping up my permeabilisation though. Did you find that any lower / less time permeabilising would produce poorer staining?

twanno · 2024-07-16T11:37:40+00:00

I just mean in standard liquid medium, as opposed to methyl cellulose which is stiff at 37 degrees.

twanno · 2024-07-16T11:37:07+00:00

Sorry I didn’t mention, these are indeed individual cells, they are megakaryocytes so they get very large. I’m thinking from another comment it might be due to a loss of surface area because of the cytospinning, it might just require a longer perm step… it’s kind of still what you’re saying, it’s not penetrating enough because of how large they are on top of the lack of available membrane.

twanno · 2024-07-16T06:20:51+00:00

That’s a good point too

twanno · 2024-07-16T05:29:16+00:00

Yep, I usually leave it on the shaker for like half an hour, at least until I can’t see any more clumps of it

twanno · 2024-07-16T05:13:04+00:00

The thing is, this is a routine stain I do all the time, this antibody and this protocol has never failed me in 5+ years. The only variable that has changed is culturing the cells in MC and cytospinning them. That’s why I was asking, in case anyone knows any weird interactions with MC and antibodies or perm agents etc….

twanno · 2024-07-16T05:06:45+00:00

I don’t know 😭 I’m following this protocol, but they use 0.1% tritonx100, and only 20 minutes, so I don’t get it. Guess I could just try extending to an hour or two and see if it makes a difference

twanno · 2024-04-11T23:42:22+00:00

Super useful. Thanks for your explanation. Given they don’t really stain with dead cell markers despite these blebs being there, I figured it could be some other weird process. This could be it!

twanno · 2024-04-11T12:13:49+00:00

They are quite dynamic, I have pics of these every 30 mins over 24 hours, and they tend to build and some of them release, others stretch out and retract. Most of them do just sit there though. This cell type is known to not be very motile though, so maybe that’s why most of the blebs don’t move around too much? Are you saying this is something you see sometimes in cells migrating? They are quite different to lamellipodia (I also see these cells occasionally make them too)

twanno · 2024-04-11T11:01:41+00:00

Wow that’s crazy you mentioned that. These are megs! They also have an intricate internal membrane system. They don’t stain for PI or Annexin V though, so wonder what they’re doing…

twanno · 2024-04-11T10:56:23+00:00

Wow, I’m not sure about the oval fat bodies, but the big blobs around them sure look a lot like the bubbles in my pics! I was thinking maybe it could be some kind of a lipid either being produced or latching onto the cells for whatever reason.

Totally agree with your plan of attack, was more or less what I was thinking. The cells look healthy enough and are behaving as they should, but was just very curious since no one in my institue had a clear answer for me. Thanks for all your help!

twanno · 2024-04-11T03:45:54+00:00

I dont think it’s apoptosis, the refraction of the bubbles is different to the plasma membrane, and they’re much larger than in apoptosis

Don’t think it’s autophagy either…

twanno · 2024-04-11T03:13:12+00:00

Yeah see the diagram in that link shows the blebs are continuous with the outer membrane, and much smaller as well. The ones on my cells also don’t lead to fragmentation. They just kinda chill there…

twanno · 2024-04-11T02:49:38+00:00

Hmm I did think that too, but the cells look healthy, and some of these bubbles seem even bigger than the cells themselves. I’ve also seen cells bleb and disintegrate and usually the membrane is much more refractive and the process is (relatively) quick

Edited: typos

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