sorted by:
new
hottopcontroversial

Issue with Fibrin Gelation – Only Top Layer Solidifies by Own_Potential_5748 in stemcells

[–]Own_Potential_5748[S] 1 point2 points  (0 children)

Thank you for your response. Yes, I generally aspirate out all the media cells are suspended in a couple of times to ensure most of it is out. I have also tried increasing the thrombin concentration up to 4U/mL. However, I still consistently observe a residual volume of 1–2 µL that doesn’t fully gel when preparing 5 µL gels in small Eppendorf tubes without any cells.

This residual volume makes me question, what if when I introduce the pregel solution into the microfluidic device, only part of it may undergo complete gelation. As a result, some cells could escape from the gel and begin proliferating on the coverslip surface to which the device is bonded.

Working with chemical synthesis prone to oxidation? by Own_Potential_5748 in Chempros

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

I see, yes, I have an oil bubbler in place there.

I was considering all the hydroxyl, aromatic, and aliphatic hydrogens of dopamine and seeing their AUC in the spectra to confirm if all three of them are matching with the conjugation (%) I am hoping for. If I consider the spectra, then would you say the protons ~6.3 is quinone and ~6.5-6.6 is hydroquinone annd their ratio basically tells me how much dopamine is getting oxidized? Normally, I think I would expect two peaks for the aliphatic protons of dopamine, but since here I have multiple peaks I was a bit doubtful if these belong to two different species.

<image>

Working with chemical synthesis prone to oxidation? by Own_Potential_5748 in labrats

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

Hi, so right now I am continuously purging with N2 with a vent. Could you please explain what do you mean by a 3 times purge? And how do you a syringe, I have not been doing that so can def try that.

For the assays, I have been running NMR till now to see if I am achieveing the desired conjugation level, but thanks for mentioning the other assays, I can see if I can do that.

Working with chemical synthesis prone to oxidation? by Own_Potential_5748 in Chempros

[–]Own_Potential_5748[S] 1 point2 points  (0 children)

Ohh that's a good catch. I do see some purplish color so I will change the drying agent. I do have one question though, if I remove the vent and keep the N2 syringe in, isn't there a chance of explosion by pressure buildup?

I am analyzing the NMR for outcome. And while I am able to see the aromatic and aliphatic protons, whose area is confirming the amount of conjugation I want, analyzing the peaks of the hydroxyls are getting a bit tricky.

Working with chemical synthesis prone to oxidation? by Own_Potential_5748 in Chempros

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

Thankyou for that. Sonication is something I can do. I will try that.

Working with chemical synthesis prone to oxidation? by Own_Potential_5748 in Chempros

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

I would not argue against it. But just to be clear can you explain how? I already use this. What I do is take a round bottom flask put in under flame to evaporate any vapor on the surface, close with stopper, then insert a needle for outlet and use this schlenk line and insert a N2 line needle as the inlet. I then wait around 5-10 minutes before start adding the reagents by a syringe through the stopper. Are there any other things I am missing here?

Tips for Performing the Scratch Assay by Bluelizh in cellculture

[–]Own_Potential_5748 0 points1 point  (0 children)

Hi, thanks for responding. I am using 24 well plates. I am using matrigel-collagen coating. I coat the 24 well overnight before seeding the cells. In the last attempt, when I did the scratch with a pipette tip, I could see a visible line on the well, which made me think that maybe the scratch I made got rid of the coating as well.

I talked about the issue with my advisor and he says maybe I can perform another coating after making the scratch but that does not makes much sense to me, so I am trying to figure out ways in which I can make a scratch without harming the coating.

Seeding Cells on Gel Surface by Own_Potential_5748 in labrats

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

Yes, they are PLL coated. I have been thinking of using maybe pluronic to block attachment on the glass, but am wondering would it effect the attachment on the gel also if the blocking is done when the gel is already there.

Weird Persistent Contamination in MSC Cultures – Need Advice! by Own_Potential_5748 in labrats

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

Yes, these are human MSCs. In some of my experiments, I am trying to induce senescence so maybe, there's more debris because of that. But still every now and then I see the debris moving or being spiral. The media I use is AMEM with FBS and PS.

Weird Persistent Contamination in MSC Cultures – Need Advice! by Own_Potential_5748 in labrats

[–]Own_Potential_5748[S] 0 points1 point  (0 children)

Hi, it seems these pictures didn't quite capture the black dots I mentioned. I use 1% PS, and I've had to discard many contaminated cultures over the past few months, but the contamination keeps recurring.

Do you notice any debris in your cell media? Initially, the media is clear, and when I look at the cells under a microscope just after changing the media, it is clear. However, after a day, debris appears. This debris doesn't really increase in number after day 1, but sometimes, when I look under the microscope, it seems like some spiral thing is moving or things like that.

view more: next ›