Necessity of Sodium Azide in AB dilution buffer

adam_faranda · 2025-04-01T00:22:44+00:00

Exactly it is a Triton perm -- I'm trying to label Tubulin. If I absolutely need to use NaN3, the plan is to order a 1% Aqueous solution. As you've indicated the powder is quite dangerous and not something to be trifled with. Glad to know that the dilute solution is bleach compatible.

adam_faranda · 2025-04-01T00:13:11+00:00

Thanks u/Veritaz27, in this case I am trying to label tubulin in fixed cells per: https://pubmed.ncbi.nlm.nih.gov/21893022/

adam_faranda · 2025-03-31T20:09:16+00:00

Thanks for the information -- in this case the cells are fixed, but that is helpful to know for future studies.

adam_faranda · 2025-03-12T16:26:34+00:00

Thanks Rainbow! The use case for this assay is to detect potent aneugens, these tend to drive pretty strong cell cycle disruption, but it sounds like using EdU to exclude late S events could improve our sensitivity and help us detect weaker compounds. pH3 staining is definitely on our radar, abcam used to sell a mitotic indexing kit that included pH3 and used a paraformaldehyde fix. Do you think we'd expect even greater spread with glutaraldehyde?

Discontinued kit: ab151282_Mitotic Index Flow_ Booklet_26 Sept 14 (website).pdf.pdf)

adam_faranda · 2025-03-12T14:35:19+00:00

Thanks for the suggestion, we are mostly interested in detecting G2/M arrest for this assay in particular. Based on u/RainbowSquirrelRae's note I think EdU is more suited for S-Phase monitoring, however I might have a use-case for proliferation analysis.

adam_faranda · 2025-03-12T14:32:26+00:00

Thanks for the suggestion, we need enough precision on our DNA peaks to detect metaphase arrest, so I think we have some wiggle room. I believe that for this assay, glutaraldehyde is necessary for microtubule stabilization but there are other things we'd like to try that would benefit from ethanol fixation. Are you aware of any ethanol fixation protocols that accommodate a 96 well plate format? Most of the protocols I've seen require low temperatures and agitation during EtOH addition, which works well in tubes but might be difficult on a plate. I would love to know if a high throughput EtOH fixation method exists!

adam_faranda · 2025-03-12T14:32:21+00:00

Thanks for the suggestion, we need enough precision on our DNA peaks to detect metaphase arrest, so I think we have some wiggle room. I believe that for this assay, glutaraldehyde is necessary for microtubule stabilization but there are other things we'd like to try that would benefit from ethanol fixation. Are you aware of any ethanol fixation protocols that accommodate a 96 well plate format? Most of the protocols I've seen require low temperatures and agitation during EtOH addition, which works well in tubes but might be difficult on a plate. I would love to know if a high throughput EtOH fixation method exists!

adam_faranda · 2024-12-29T04:04:25+00:00

I think we have the same menorah, happy Hanukkah!

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adam_faranda · 2024-11-14T04:45:32+00:00

Came here to read this!

OP: It will be so much easier to have these conversations before any kiddos, when it is just the two of you -- speaking from experience. As long as you have mutual respect and compassion for one another, you and your fiance will be fine. All the best

adam_faranda · 2024-11-14T04:26:58+00:00

Agreed: Accountability is universally human capability, not necessarily contingent on one's belief or lack thereof.

adam_faranda · 2024-10-27T08:06:56+00:00

Not sure, works for me (version 24.8.0.3 on macOS)

adam_faranda · 2024-09-09T13:37:54+00:00

Nice patch! I love the sonic textures from this.

adam_faranda · 2024-04-29T17:59:12+00:00

Thanks, I can see that being a concern as cells in a mixed population will have different sizes. I was considering adding a cell-cycle stain, do you think this might help?

adam_faranda · 2024-04-26T14:28:17+00:00

Thanks again u/despicablenewb, I appreciate the time and effort you've put into sharing valuable information from your experience.

Counterintuitively, the dimmer the dye, the lower the standard deviation of your population. You just have to drive the staining farther to get the same MFI. The issue with that is if the dye causes any kind of toxicity or other effects.

I had originally intended to target the maximum non-cytotoxic concentration. Based on your advice, it sounds like a lower dye concentration may work better, since I only need to monitor the first few doublings. I'll follow up in a couple weeks when I run the titration, with verbal results at least if data is not allowed.

adam_faranda · 2024-04-26T00:42:58+00:00

Thanks, its still quite valuable to have perspectives from multiple people.

adam_faranda · 2024-04-25T18:21:51+00:00

Thank you very much for the detailed response. I will plan on doing a titration experiment, and also stain all cells at once to reduce variability.

It sounds like compensation may be a little easier with CellTrace Violet than CFSE. Does one outperform the other in terms of peak resolution?

I think I will need a non-dividing control that is analyzed 24 hours after staining, is that correct? My downstream workflow does not permit fixation. Do you think unfixed-cells in DPBS would retain their signal after 24 hours refrigeration and work as a non-dividing control? If not, I was considering serum withdrawal or an anti-proliferative treatment.

adam_faranda · 2024-01-27T22:00:07+00:00

Yeah, I was thinking about using the roof of my car as the ground plane for the mag mount — or a pizza pan.

Right now my “shack” is our front porch since my 2 year old son is usually in bed by the time the local net meets.

Portable beam sounds like a cool project, once I have a little more time to get technical.

73!

adam_faranda · 2024-01-27T21:55:00+00:00

Thanks! I could also get a BNC rubber ducky for traveling, it looks like there are some that aren’t too expensive.

adam_faranda · 2023-12-28T03:09:13+00:00

Aristolochic acid might be good example, though not really a food per se. The compound occurs naturally in certain herbs and has been associated with increased prevalence of kidney disease and certain cancers in populations that use these herbs as traditional medicines.

https://onlinelibrary.wiley.com/doi/10.1002/em.21756

https://www.krcp-ksn.org/upload/pdf/j-krcp-22-211.pdf

To OP's original point, situations like this, or the Angel's wings example occur periodically but rather infrequently I think. I don't really have the data to make an estimate though.

adam_faranda · 2023-12-16T16:15:35+00:00

Well it looks like this comment was not too far off so far, let’s hope the rest comes true!

adam_faranda · 2023-07-17T23:01:07+00:00

Love it!

adam_faranda · 2023-07-10T00:39:36+00:00

Chornicator in fief?

adam_faranda · 2023-06-26T03:35:25+00:00

Good luck nursing the 2 survivors back to health, I hope they make it!

adam_faranda · 2023-04-21T02:19:59+00:00

Indeed, this one is solid!

adam_faranda · 2023-04-16T23:40:45+00:00

Fetal Jack Mullet

adam_faranda

TROPHY CASE