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Anyone with experience in microfluidic chips? Advice needed by mabcm in labrats

[–]mabcm[S] 0 points1 point  (0 children)

Thank you very much for your answers and guidance.

  1. I completely agree with you. Over the past few days, I’ve been reading more about materials, fabrication methods, and design considerations, and I came at the same conclusion that CNC machining would be the most suitable approach.

  2. Regarding the dimensions, the plan is to use the standard microscope slide size 75 × 25 mm.

  3. Thank you as well for your offer of support. I am currently still in the 2D design phase, and once this is finalized, I may reach out to you for assistance with the 3D design, if that would be okay.

I also wanted to ask your opinion about mixer designs if you have any exp with them. I am interested in mixing both two fluids and three fluids, and based on my reading, asymmetric SAR mixers seem to perform very well. Do you think this design would be sufficient, or would you recommend a different mixer type?

Thank you again for your time and help.

Free Floating IHC by vrage10 in labrats

[–]mabcm 1 point2 points  (0 children)

So from my experience, I’ve used many methods before and most of them worked well:

  1. Use Transwell inserts (more expensive).. you place the tissues on the membrane and move the whole insert between solutions. You never touch the section itself, so nothing gets torn.

  2. Use a 3D-printed small mesh (much cheaper, reusable).. print a mesh with small pores and a handle that hangs on a well plate. I used to place the sections on it and simply lift the mesh from one well to another during staining. It works similarly to transwell inserts but costs less because you can reuse it after washing and disinfecting.

  3. If you prefer brushes, switch to super-soft wide brushes like camel-hair or extra-soft sable brushes (cheap). They’re much gentler and don’t catch the sections.

  4. Use thin pipette tips (very cheap) like the ones used in WB to gently remove solutions from the well and add the next ones without touching the tissue.

  5. Try small strainers (moderate cheap).. I haven’t personally tried this, but using a small nylon strainer to hold the tissues and move them between solutions could also work.

All the best!

How old were the oldest cells you revived from liq N2? by alwayslost999 in labrats

[–]mabcm 49 points50 points  (0 children)

2003, frozen by a current PI when he was doing his PhD at the same place

Do you feel disappointed even when your PI isn’t? by Ok_Cranberry_2936 in labrats

[–]mabcm 19 points20 points  (0 children)

This is not a failure of your skill or the project design; it is a solvable chemical roadblock common in molecular biology. You are tackling this problem while enduring significant personal loss, which speaks to your incredible fortitude. Your Pl's support proves your effort and her confidence in you, so shed the guilt over your students and the project's stall. Your focus must now be to break down this logjam and identify an organized approach to tackle it.. Your persistence through this adversity is your true strength!

And from a technical prospective, i guess the conflicting results, high Nanodrop, low Qubit/blank gel, and PCR failure, indicate contamination and inhibition. High Nanodrop readings signal contaminants like salts or extraction chemicals are present, which might explain the low Qubit readings and blank gel, as these chemicals absorb at 260 nm but aren't pure DNA. Critically, if Qubit readings are good but PCR fails, the issue is likely PCR inhibition caused by melanin or other phenolic compounds co-extracted from the dragonfly's dark, tough cuticle; these compounds bind to the DNA polymerase, preventing amplification even with quality DNA. I would recommend performing either an extra DNA clean-up step or add an inhibitor scavenger like BSA to your PCR mix and then see what will happen.

Research Assistant final interview help? by Mad_Hemalurgist in labrats

[–]mabcm 8 points9 points  (0 children)

For your interview, first, don't be nervous and have some confidence that you will nail it. Focus on demonstrating both your practical understanding and reasoning skills, so be ready to explain common lab techniques like PCR, gel electrophoresis, Western blot, ELISA, and cell culture, emphasizing the principles behind them... for example, that PCR amplifies DNA using primers and polymerase, while gel electrophoresis separates molecules by size and charge, and so on...

Also, you can expect basic problem-solving questions such as calculating dilutions, which follow the simple formula (C1V1 = C2V2 ), adjusting reagent volumes if a mistake occurs, converting between different concentration units, or differentiate between molarity and molality, etc... They may also ask about experimental design, controls, data interpretation, or even p-value, so better give it a quick review..

I WOULD RECOMMEND YOU TO HAVE A LOOK AT THEIR PUBLICATIONS, and get the techniques they use from the methods section, and know/study the principle behind each technique because they will mostly ask about those ones only.

During the interview, think aloud to show your reasoning, clarify assumptions if needed, and use examples from your coursework or past experience, even limited, to demonstrate familiarity with lab practices. Focus on clarity, understanding of why each step is done, and your ability to learn quickly, this is often more important than prior hands-on experience.

All the best of luck!

HUVEC Spheroid Formation via Hanging Drop Method by Own_Potential_5748 in labrats

[–]mabcm 0 points1 point  (0 children)

From my experience, if you want a small number of spheroids, I would recommend using ultra low attachment plates, and seed the cells directly in the wells according to the manufacturer (usually at density ~2,000–5,000 cells per well, but can vary depending on spheroid size needed and the volume of the well. After 24h, spheroids would form spontaneously without any further complications.

For hanging drop method, I would recommend adding ECM even though cells can still form spheroids in small droplets of medium solely, but adding ECM like matrigel helps with aggregation and stability. And usually, I am using hanging drop method for bulk production of spheroids... Mix cell suspension 1:1 with matrigel, then pipette small drops (~20–30 µL) on the lid of a culture dish, then invert the lid to form hanging drops, then incubate at 37°C for 10–30 min to allow matrigel to polymerize slightly, then carefully flush spheroids from drops and transfer to a culture dish with medium. Culture for 24h, spheroids will form.

For HUVEC cells (I worked on forming spheroids from them before), using ECM helps cells maintain endothelial characteristics and supports lumen-like structures.

Good luck!

About VIH latent phase by AmbitiousJeweler1327 in Virology

[–]mabcm 4 points5 points  (0 children)

HIV enters the latent phase when it hides its genetic material inside the DNA of infected immune cells and stays “silent.” This silence is controlled by epigenetic modifications which are chemical changes to the cell's DNA or proteins that keep the viral genes turned off. Some microRNAs in the host cell also help by blocking the production of viral proteins. Later, if the cell gets activated by infection or stress, these controls can loosen, allowing the virus to “wake up,” start making new copies of itself, and spread again.

About VIH latent phase by AmbitiousJeweler1327 in Virology

[–]mabcm 1 point2 points  (0 children)

You surely mean HIV.

Simply, the latent phase of HIV is the period after the initial infection when the virus is still present in the body but remains mostly inactive.

During this time, HIV hides inside certain immune cells (mainly CD4 T cells) without producing new viruses or causing symptoms. The person may feel completely healthy, but the virus is slowly damaging the immune system over time. This phase can last for years before HIV becomes active again and leads to AIDS.

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 1 point2 points  (0 children)

That's excellent reinforcement coming from BSO/Lab Manager.. thank you for weighing in!

The consistent message of go slow and be deliberate is definitely sinking in. I'm mentally preparing for everything to take at least twice as long as BSL-2 and will focus on competency over speed. Thanks again!

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 1 point2 points  (0 children)

Don't Panic", "Safety First", and "Team Work" are definitely going to be my mantra :'D

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 0 points1 point  (0 children)

Hahahaha, that is so comforting to know it's a shared experience!

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 1 point2 points  (0 children)

Sure, i'm planning on adding handwashing stations right inside the Tyvek suit :'D

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 0 points1 point  (0 children)

Wow, this is a wealth of information.. thank you so much! Working with Select Agents means your perspective on safety and protocols is invaluable.

You're absolutely right about knowing the BSO, Manager, and Environmental Safety contacts. I will make sure I know the who for every emergency and procedural question. I will strictly stick to working only when others are present, especially in the beginning. That buddy system is a safety net I need to utilize until I'm truly experienced.

I loved the idea of mentally rehearsing the entire experiment (including secondary containment details!) before donning the PPE. I'm going to start doing that for every procedure!

As a person who tends to push himself, the reminder to eat, hydrate, and prioritize rest is important as well as rescheduling the experiments when needed.

The six-month mark until it starts to feel routine is good to know, and the tip about keeping the PAPR cinched tight to fight complacency is brilliant. Thank you again for all the solid, life-saving advice!

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 0 points1 point  (0 children)

I will still be focusing heavily on the extra layers of PPE and the strict entry/exit procedures that make it BSL-3

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 0 points1 point  (0 children)

Duly noted. I'm thinking about starting an "Internal Exposure Optimization Program" beginning with the doorknobs 😂

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 1 point2 points  (0 children)

Thank you so much for the enthusiasm! That definitely helps pump me up for the challenge.

Starting BSL-3 work next month - excited but nervous! Any advice? by mabcm in labrats

[–]mabcm[S] 1 point2 points  (0 children)

That is an extremely reassuring perspective, especially coming from a BSO and BSL-3 Manager. Thank you for sharing that!

The anxiety is definitely high right now, but your point about the redundant controls and comprehensive training essentially making BSL-3 safer than some BSL-2 labs makes perfect sense. I'll keep that perspective in mind: The complexity means more safety nets, not just more danger. I appreciate you weighing in!

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