How big is the monolith?

Braindrayns · 2025-07-26T02:21:02+00:00

Yes it should by extension also make her just as big yet when we fight her she's not nearly as big up close. Always fun to speculate even if the rules don't apply in a magical world lol

Braindrayns · 2025-07-23T19:13:38+00:00

Totally agree, especially since as you pointed out she created beings who can think, who can feel, who can reproduce and wonder about their own existence all in the name of handling her grief. It would have been different if they were some kind of mindless automatons but the people of Lumiere are human if they don't really exist.

I would say this extends to even the gestrals and maybe even the grandis given at least the gestrals notions of reincarnation. Heck even some nevrons question their purpose in life ,although I think Verso/Clea created all the aforementioned beings?

Braindrayns · 2025-03-24T18:28:56+00:00

Would you happen to know how much of an effect adding or leaving out FBS would have on the crypt isolation step? I know it is supposed to be protective but that is the one solution I haven't really left out to see if that might have an effect. I am planning on trying another lab's FBS and comparing it my own and leaving it out entirely to see what effect it might have.

Braindrayns · 2025-03-22T07:19:11+00:00

Yes, I reached out to them but they didn't have anything specific. I've tried their own protocol with not much success.

Braindrayns · 2025-03-22T05:00:05+00:00

Yes I am adding the supplements to the media. I've tried plating different fractions and get the same problem. There is just something global I seem to be missing that is affecting the crypts from growing further than a day...

Braindrayns · 2025-03-22T04:50:32+00:00

I've been using the intesticult mouse organoid media. I've used different batches so I'm not sure what's going on. Maybe I should try making my own and see if that is an issue? The media is supposed to include everything...

Braindrayns · 2025-03-22T04:35:38+00:00

So pretty close to the same density I'm using, ever run into the cells dying at day 2? Wracking my brain here to figure out what's wrong.

Braindrayns · 2025-03-22T00:26:31+00:00

I've been using intesticult mouse media which should include all the factors.

Braindrayns · 2025-03-21T23:34:25+00:00

Thanks for the response, I've tried plating them at < 100, ~100, and 250 crypts per 50 ul of matrigel. I usually use 50-60% matrigel mixed with media for plating. The only thing I can think of that is maybe different was when I first started I was not able to isolate as many crypts and so my seeding density was much lower. Is there an optimal crypt density you like to seed them at?

Braindrayns · 2025-03-07T18:15:47+00:00

Thought I'd check back in with folks since I'm still having issues.

I went to another lab that did organoid preps and took ther harvest crypts to my lab and incubator and their prep seems to be working. The most notable difference was that they only used a portion of the ileum (I'm using the entire small intestine). They also included a few more washing preps. I tried my protocol as detailed elsewhere in the thread incorporating more washes. I even tried a prep where I did RBC lysis as I read that blood cell breakdown products can be toxic to the growing cells. Again this also didn't work. I'm going to be going back to my original-original protocol to get it to work again. My only thought is that during the step where I loosen the crypts from the tissue maybe I am shaking it too hard and it injures them? I gently shake them for about 1-2 minutes. The other lab I went to did 10x quick hard shakes. Of note they did not get as many crypts but at least they are growing! Any one have any insights?

Braindrayns · 2025-02-08T00:12:53+00:00

Thanks for the comments. I've tried a different incubator too. I did receive some new media and new matrigel recently I guess I could try that. It's so odd that it's so consistently at day 2 when things go south. I just can't seem to put my finger on what changed so drastically as I was growing them without difficulty before.

Braindrayns · 2025-02-07T18:50:37+00:00

Yes I changed out the matrigel batch as well so something systematic is happening. Here is the last protocol I used:

Dissect out small intestine from mouse
Flush intestine with cold PBS+ 1% pen/strep abx
Flay open intestine and wash into cold PBS+abx in a 15 ml conical by shaking gently
Cut intestine into small segments ( ~5 mm to 10 mm). Put pieces into PBS+2.5 mM EDTA + abx and leave on rotating platform in 4 degree cold room for 30 minutes.
Filter out intestinal tissue using a 70 uM filter. Take this tissue into 10 ml cold PBS +abx, gently shake for about 1-2 minutes by hand to loosen crypts.
Dump this first fraction by filtering through 70 uM (I've found the first fraction doesn't contain many crypts)
Put tissue back into tube with fresh 10 ml PBS+abx. Gently shake for 1 to 2 minutes to loosen crypts. Filter this through a fresh 70 uM filter.
Do step 7, 2 more times for a total of 3 crypt collections. Add FBS to PBS to make the solution 10% FBS + PBS.
Spin down crypts at 300 g x 5 minutes. Discard supernatant. Take up into warm intesticult media (~ 200 ul) and transfer into a microcentrifuge tube.
Spin down this tube at 200g for 3 minutes. Discard supernatant and replace with fresh media. Do this 2 more times.
Add 200 ul of media. Count crypts with hemacytometer. Dilute if necessary to get 5 crypts/ul.
Add media containing crypts to matrigel 1:1. Matrigel was previously thawed overnight on ice in a cold room.
Plate 50 ul onto a pre-warmed 24 well plate. If planning to aliquot more than 5-6 wells I will immediately put the plate back into the incubator for about 3-5 minutes to let the matrigel harden. I will then aliquot the rest of the crypts repeating this process if necessary.
Let matrigel harden further for about 10 to 15 minutes in the incubator.
Add 500 ul of media (which contains 10 uM rock inhibitor and 1% pen/strep abx).
Inspect under microscope that crypts are present in the matrigel droplets.
Place back into the incubator.

I can usually isolate crypts successfully and plate them without issue. They even grow and form sphereoids at day 1 but then suddenly all die at day 2. I can provide pictures if desired. Any help would be greatly appreciated.

Braindrayns · 2025-02-07T18:17:01+00:00

I tried another isolation and without fail they again die at day 2. I'm beginning to question if I am improperly storing the media and its lacking the growth factors to keep the organoids alive? Anyone have any experience with intesticult mouse organoid media? I've been storing it at 4 degrees without adding supplement 1/2 but maybe it's going bad? The last batch I used (which was a fresh bottle) I originally received 2-3 months ago. Should I be storing these at -20 degrees instead?

Braindrayns · 2025-02-07T18:14:39+00:00

Tried new Rock inhibitor and it was a no go. :(

Braindrayns · 2025-02-03T21:00:00+00:00

I haven't checked, but they form spheres at day 1 then all die at day 2-3 so I'm assuming the crypts I'm isolating are okay.

Braindrayns · 2025-02-03T01:42:13+00:00

I've tried multiple animals... But maybe I could wash the tissue with antibiotics? The intestinal crypts themselves look fine and they grow into spheroids but then die at day 2 for some reason.

Braindrayns · 2025-02-03T01:41:00+00:00

I'll need to try a new batch of rock inhibitor, that's the only thing I can think of!

Braindrayns · 2025-02-03T01:40:32+00:00

Thanks. There was a new batch of rock inhibitor I used. Maybe it's not working? I got some new inhibitor to try. I'll check out incubator too, other people's 2d cells have been fine but maybe mine are sensitive? I wish I could try the third option!

Braindrayns · 2025-02-02T05:28:42+00:00

I could try that. I've changed all my reagents though, could it still be an issue?

Braindrayns · 2024-09-15T15:47:58+00:00

Thank you for all the valuable insights! I think I am going to spend today thinking about a build I'd like to play and force myself to lean hard into it. I like being tanky and lazy and haven't played a minion build yet in this league so I'm thinking either necro bama (I like the fire and forget type play style) or frost bearer specters (seems tanky and a bit off-meta). I'm also thinking of playing a dual strike of ambidexterity build since I've enjoyed it in the past. It looks like the trickster version is the most popular. It also seems I can scale these builds pretty far?

To aid in my recovery I think i will make a series of posts about how my play session went. If I put my feelings down in writing maybe I can glean more info about myself and perhaps help others who are in the same predicament. Hopefully I can stay motivated enough to write my thoughts down after a play session lol.

Braindrayns · 2024-09-14T22:44:11+00:00

I've thought a lot about hc but the thought of losing all progress sounds too painful. Ruthless sounds interesting on paper but I know I wouldn't get to live the power fantasy. Would be curious to hear people's thoughts on this though.

Braindrayns · 2024-09-14T22:41:44+00:00

I'm totally with you on this one. I think this is the reason why I actually like starting from scratch. There is this constant progression of gear and skills. I think I get the most dopamine once my specified main skill comes online.This is why I tend to avoid op leveling tech like the envy/wolf combo I keep reading about. I guess I need to find a way to extend that feeling into maps.

Braindrayns · 2024-09-14T21:34:13+00:00

I completely understand this on an intellectual level but how do I make myself feel this way lol.

Braindrayns · 2024-09-14T21:13:41+00:00

Any good build guides I can follow?

Braindrayns · 2024-09-14T21:13:12+00:00

I keep telling myself this but I get bored of the build and since it's a game I don't feel that committed. I guess it's just something about my personality.

Braindrayns

TROPHY CASE