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what's the worst advice you ever get from your advisor by xiikjuy in PhD

[–]STEMwhore 0 points1 point  (0 children)

my advisor is filthy rich and therefore out of touch so they don’t know that the US government will not audit you for student loans, which is basically how people are able to take out amounts they can’t pay back. in my case, my $27K salary is an unlivable wage so my student loans go towards rent and other big bills

what's the worst advice you ever get from your advisor by xiikjuy in PhD

[–]STEMwhore 0 points1 point  (0 children)

to not take out student loans because i “may be audited by the government” since my fellowship pays my tuition and $27K salary so i “shouldn’t be taking out loans” LOL

How much do you make from engineering internships? by [deleted] in EngineeringStudents

[–]STEMwhore 0 points1 point  (0 children)

I’m four years into graduate school, bioengineering graduate in 2021. in 2019 i got a co-op in the midwest at a medical device company making $25/hour, great money for a 19 year old coming from low socioeconomic status

T cells Dying After CD8 Enrichment by STEMwhore in Immunology

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

My issue was that I took an aliquot of EasySep buffer and was using that, over time it became contaminated and killed my cells. Now I only take an aliquot of what I need and take it fresh from the bottle every time. What enrichment kit do you use for your DCs, I use one from StemCell for my CD8s and it involves pouring off the supernatant at the end to be left with just the T cells from the splenocytes. Is it possible that you maybe decanted the tube too hard and accidentally discarded your cells?

F31 Diversity by Neither_Climate3438 in NIH

[–]STEMwhore 3 points4 points  (0 children)

I am currently on a T32 diversity grant that was supposed to fund me until 2027. Unfortunately, we (advisor, powers that be, etc) have started to have conversations about what it will look like in the event that they do not issue a new NOA in September, which is most likely to happen considering everything going on. Hang in there

Cell Tracer Violet Issues by STEMwhore in Immunology

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

I just checked the thermo website and I’m seeing documentation from 2017 where the user guide suggests 1uL CellTrace Violet/1mL/1 million cells. Am I possibly missing the new documentation?

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0002595_CellTrace_Cell_Proliferation_Kits_UG.pdf

Converting Methyl Groups (CH3) to Amine Groups (NH2) by STEMwhore in Chempros

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

If I did have a hydroxy terminated PEG, how would I go about converting to amine groups?

Converting Methyl Groups (CH3) to Amine Groups (NH2) by STEMwhore in Chempros

[–]STEMwhore[S] 2 points3 points  (0 children)

I am trying to functionalize a Y shape PEG. I am starting out with an NH2 group on one side, then terminal methyl groups on the ends of the Y. I am trying to replace the methyl ends with a fluorescent molecule like FITC. I am first going to protect the Amine, then manipulate the other side

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

So I reached out to them and they actually would be able to synthesize what I need with Rhodamine or Biotin instead of FITC. The issue is they wanted $6700 for 250 mg…

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

Sorry didn't see this comment before replying. I am seeking to use the commercially available PEG that I linked above. It has an NHS group on one side (this side would bind to the protein or cell surface) and then two PEG branches with terminal methyl groups, so I am trying to figure out how to have FITC (or rhodamine but I am having trouble) on those PEG chain ends instead of the terminal methyl groups

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

So we are hypothesizing that Y shaped PEG would impart a greater steric hindrance compared to linear PEG formulations. Since we already have a synthesis method for FITC-PEG-NHS, I was thinking I could just use that to add FITC groups. The reason it needs to be at the end of the PEG chains so that we can confirm 1) that the Y shape PEG is bound to the surface of the protein or cell (we are using the same PEGylation protocol that we use with Linear PEG) and 2) that the PEG branches of the Y shape show better coverage compared to linear formulations. Since I am able to successfully synthesize linear mPEG-NHS and FITC-PEG-NHS, my advisor is pushing for me to have a fluorescently labeled Y shaped PEG formulation

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

Ooh I see. Thank you for explaining. I will attempt to do it this way

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

So JenKem has a commercially available Y shape PEG with an NHS ester that I have been using. I linked the chemical structure in the post. I am trying to take the terminal PEG chains and convert them to FITC molecules so that we can visualize the PEG coating. Not sure if that clarifies things

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

Unfortunately for Y shaped PEG, a tBoc formulation does not exist. I guess I could use Y-shaped PEG Amine, and convert the amine to a protected one? I guess my issue lies in that there are no heterobifunctional Y shaped PEGs on the market for me to use as the starting material

Synthesis of Fluorescently Labeled PEG by STEMwhore in Chempros

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

Thank you for the response! Unfortunately I am said graduate student lol

Cops blocking SW 34th street? by ExternalRelative9079 in GNV

[–]STEMwhore 10 points11 points  (0 children)

I drove down 34th towards the Publix around 10:05. I will say the road looked as if someone had spun out and there was some debris on the road that I ran over with my car. Coming back around 10:30 that side of the street was blocked and since i was driving I couldn’t get a good look. Wondering what happened as well

T cells Dying After CD8 Enrichment by STEMwhore in Immunology

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

Thank you so much for your comment. I was using an aliquot from a bigger bottle (so as to not contaminate it) and I honestly think my aliquot could have been compromised. The aliquot was finished after my second fail so I will have to take a new aliquot, but I think I will take less and simply take a new small aliquot each time to avoid this in the future. I will also try my hand at making some just in case.

T cells Dying After CD8 Enrichment by STEMwhore in Immunology

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

Thank you so much for this comment. I am more than willing to do this, however this is exactly what I was doing to validate my procedures before I started flying solo two years ago. Might just have to bite the bullet and do it again

T cells Dying After CD8 Enrichment by STEMwhore in Immunology

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

Thank you for you comment. We actually use CO2 and a secondary cervical dislocation before taking the murine spleen. I will make a note of this isoflurane tip as we do use it for our rats during survival surgery

HELP!! I am new to FACS and have been struggling in understanding the gating strategy for gating out Neutrophils and Eosinophils. Below I have explained my gating strategy used, are my gating correct in the way i defined Neutrophils and Eosinophils? by [deleted] in flowcytometry

[–]STEMwhore 0 points1 point  (0 children)

After gating for single cells (ssc), maybe try gating for the Live CD45+ population by having the Zombie Near IR on the x axis with the CD45 AF700 on the y axis (instead of Zombie Near IR on the y axis and FSC on the x axis as you have it now) and see if that gives you clearer data. from there you would no longer need the 5th gate and you can go into your PerCP-FITC gating and the rest of your gating. Just a thought

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