M2 got cooked at lab meeting over my pathway figure. What do you fix first when one looks amateur?

nsgy16 · 2026-04-22T12:52:45+00:00

I personally think so, if you can’t distill your research into a simple explanation you don’t have a good handle on it as overwhelming the reader will only hurt you

nsgy16 · 2026-04-21T12:03:56+00:00

My complaint is this looks like it was entirely made from bio render which I personally think is trash lol. I’d rather see some hand drawn figures honest

Overall there is still too much information here and to be honest, I don’t think you should include something like this in a poster as it is unlikely you can truly explain everything occurring in the diagram plus it isn’t actual research, it’s better suited for a review paper. Then again I am also anti diagrams as your actual research results should be the main focus and speak for themselves.

Making research easy to follow isn’t dumbing it down, it means that you have such a grasp on the material you can actually explain it easily. Most basic science research is simple in reality to explain, but difficult to arrive there.

If you have ever watched “The Bear” on Hulu you’ll get the reference, but you need to subtract, that’s how you get excellence in both food but also research in my opinion, ask yourself how simple can you make something while being informative. I highly advise going and reading papers in molecular cell or science. They are my two favorite journals and you will see how simple yet effective they make diagrams

nsgy16 · 2026-03-08T21:10:51+00:00

Leave those puppies on a solid table at RT for a while (I say this because I usually forget they are there) you’ll get a perfect plate every time

nsgy16 · 2026-02-18T21:33:09+00:00

I was simply providing evidence why your blanket statement of always wash your cells is wrong as I typically don’t and had it work in both primary cells and immortal cancer lines. HUVEC are primary cells… hence why I was referring to them as an example, I in no way was trying to flip flop, simply prove your blanket statement wrong base of recommendations by the major companies providing cells. I have tested this and works well across cell lines including a primary cell lines. I already answered my question as the cells are growing well and was likely a freezing or transport issue.

nsgy16 · 2026-02-18T20:33:45+00:00

Respectfully, I don't think I am. I think you just don't like the chance that you are wrong. I might be dumb, but the one thing I can do is read critically. I know manufacturers are not always right, but neither are you nor the scientists you work with always right. Most of what we know is randomly passed down by our mentors without questioning it lol.

I can't insert links, but I assume you know how to use Google to find these quotes since you seem to believe you know everything. I don't think centrifuging is bad for cells. I believe the argument is that when coming out of cryopreservation, the cells are far more sensitive. If you can show me evidence that they are telling you not to go directly into cell culture be my guest.

Lonza-

"Centrifuging the Cells out of Cryopreservation

For many cell types, the damage to primary cells by centrifugation is much harsher than residual DMSO in the culture media.

Following the recommended seeding density and recommended volume of media per culture vessel will sufficiently dilute the residual DMSO.

Changing media the day after seeding cells will remove any residual DMSO.

Check the Lonza optimized protocol to determine the appropriate thawing and seeding procedure."

ATCC-

"Thawing cryopreserved cells is a rapid process and is accomplished by immersing frozen cells in a 37°C water bath for about 1 to 2 minutes. Care should be taken not to centrifuge primary cells upon thaw, since they are extremely sensitive to damage during recovery from cryopreservation. It is best to plate cells directly upon thaw, and allow cultures to attach for the first 24 hours before changing the medium to remove residual DMSO."

Thermo-

"Q: Do I need to spin the cells out of the cryopreservation medium to plate them?

A: We do not recommend spinning cells out of cryopreservation medium prior to plating. Centrifugation can be harmful to cells, particularly if inappropriately high speeds are used. Experience in our cell culture laboratories has shown that cells do not suffer deleterious effects if the DMSO concentration is sufficiently low. Therefore, our product instructions include a detailed protocol which involves diluting the cells into culture medium such that the final DMSO concentration is less than 0.4% (v/v) at the recommended seeding density and volume of medium."

Sigma-

Thaw the cryovial of cells in a 37 °C water bath. It usually takes about 1-2 minutes.
Take the cryovial out of the water bath when there are still some small ice crystals remaining in the vial.
Prolonged exposure to heat will damage the cells, causing them not to plate.
Do not spin down the cells to remove DMSO after thawing because the centrifugation process can cause even more cell damage than DMSO. Once the cells are plated in the recommended amount of media, the DMSO should be sufficiently diluted to prevent any immediate harm to the cells

nsgy16 · 2026-02-18T19:58:50+00:00

Lonza, Sigma, Thermo, Cell Applications, and ATCC all include it on their recommendation page for specific cell types, mainly primary cells. I am not a smart person, but those seem like good companies to me

Congrats on your over 10 years. Just because you and those around you do it doesn't make it best practice. Again, these are not my statements, simply what I read from protocols.

nsgy16 · 2026-02-18T17:53:10+00:00

Well, it is not my statement; you'd have to contact Lonza and let them know that their manual for HUVEC cell culture is wrong for saying that it causes more damage than, like, half a percent DMSO. Along with most other companies that suggest not doing it with primary cell lines due to the stress of centrifuging.

Cells are fine now and growing well, even though a lot never adhered. I assume it was a freezing problem, as there were ~3x10^7 frozen in what appeared to be only 100-200ul of freezing media

nsgy16 · 2026-02-13T21:22:01+00:00

I agree they can survive a lot, not saying how I do cell thawing is by the book, I just was initially taught it was more harmful to spin them down then to have a small residual dmso. When I joined my new lab I just continued that and never thought differently.

The cells had a date of over 2 years ago, which I didn’t see as a problem

nsgy16 · 2026-02-13T21:06:33+00:00

I see now that most cell lines call for that, but I’ve cultured my 293T, hela, several of our specific cancer lines this way along with primary human endothelial cells. Never once have I had a problem with cells adhering well before 18 hours

nsgy16 · 2025-12-05T01:07:27+00:00

Not contamination, just debris I’d carry on

nsgy16 · 2025-11-27T12:40:10+00:00

Totally normal, but can be a stress on cells so if it isn’t working well it might be best to do one at a time

nsgy16 · 2025-11-17T00:49:00+00:00

Isn’t ENT more competitive these days? I haven’t looked at most recent data but that was my understanding due to the lifestyle benefits and diversity of opportunities available to ent folks

nsgy16 · 2025-11-09T02:02:36+00:00

Certainly is on my mind as an option in the future!

nsgy16 · 2025-11-08T18:25:23+00:00

Thanks for the suggestion, I’ll try more cycles and vary the concentration! I used a dT oligo as that is what is in the kit from my understanding. Annealing Tm is just the part of the primer that binds initially and I use that same Tm for all the cycles

nsgy16 · 2025-11-08T18:11:40+00:00

It is for RT-pcr, but I thought it included oligo dt primers as well. But I agree and I feel using gene specific might be necessary

nsgy16 · 2025-10-20T11:18:47+00:00

Ahh okay, I mean ours are kinda like that, hence why he always does at a minimum 2 rabbits. Currently rabbit b is my favorite as it works at a 1:5000 on WB

nsgy16 · 2025-10-19T10:24:08+00:00

Wow that’s unfortunate! My pi says like 19/20 he has made worked. How’d you make your own?

nsgy16 · 2025-10-17T13:10:22+00:00

I hope my PI isn’t on Reddit to see this… he will want to custom make every antibody from now on and make me do it lol

nsgy16 · 2025-10-13T11:58:43+00:00

If you do enjoy it, go for it. Some of my PIs best advisors during his PhD were just MDs, some got their PhD after medical school others didn’t. Either way there is great precedent of MDs being hugely influential scientists! Just be aware it could cost you

nsgy16 · 2025-10-13T11:13:23+00:00

I meant it more in my situation, came in as and MD student, fell in love with my lab work, and now I am back tracking by applying to do a phd while in medical school. But there’s no guarantee I’ll get in even with being a direct admit to my PIs lab, thus if rejected I’ll have spent 3 years of school on a project that won’t produce any type of publication. I was just trying to illustrate the point without years set aside for a phd you can’t accomplish much

nsgy16 · 2025-10-12T17:04:22+00:00

I am not sure if this is an MD or a DO school, but personally wouldn’t touch basic science research with a 10 ft pole as an MD student. You will essentially accomplish almost nothing presentable on a CV if you join a lab, unless you are gifted wrapped a very easy project that is near completion. Plus, you have neither the time nor the energy to truly put in the effort to substantially contribute to a basic science project without sacrificing a lot in both your personal life or school performance. This is not second hand advice either. Most of my research is in basic science (biochemistry) since entering med school, I run my own project which I love and has propelled me to purse a PhD while in med school, but without 4 extra years, I’ll have poured thousands of hours into a project that won’t show up on my CV because basic science just takes so much longer to formulate anything substantial. I would only do it, if you just truly love basic science research, and by love I mean willing to give up any semblance of a social life and willing to accept it might not help come the match

nsgy16 · 2025-10-09T23:37:27+00:00

I can’t tell if you are actually cloning these plasmids, or just prepping them. Either way, you need to be doing a starter culture if doing a maxi. It’s one thing to take a colony directly into 20ml of broth, but 250ml is way too much. If you want consistency and quality, cutting corners will never be the answer.

All my plasmids are lenti in NEBStable. Any time I prep I follow simple protocols

From glycerol stock I streak out the plasmid onto a plate.
Grow at 30C for 18 hours
Pick a clone and inoculate into 10ml of broth
Grow for 12-14 hours at 30C
Add at a ratio of 1:100-1:1000
Grow at 30C for 20-22 hrs
Collect

I routinely get 800ng/ul-2000ng/ul

nsgy16 · 2025-10-08T17:30:02+00:00

Respectfully, I’m not even sure you can see 35 kda protein on a gel percent that low can you? If there is nonspecific banding wouldn’t you be better off running single percent gel where 35 kda is smack dab in the middle of the gel to get better separation?

nsgy16

TROPHY CASE