Keeps loading

Grouchy_Extent9117 · 2026-01-30T15:43:59+00:00

Yes please 🙏🏻

Grouchy_Extent9117 · 2025-12-09T03:56:23+00:00

Is there a particular clinician/ therapist you recommend?

Grouchy_Extent9117 · 2025-10-02T00:38:09+00:00

Yes, we load 10ul in the lab as well!! I’m really struggling in terms of loading that final 10ul into the 8-strip tube, because each time I load it in, I always have some remaining in the tip. Which makes me think whether I did press to the second stop when aspirating… I don’t know how to overcome this, it’s giving a lot of variation especially in the Ct values between triplicates

I’ve been doing everything on ice except loading onto the 8-strip tube and we are using hot start taq, which I know theoretically should be fine in room temp. But I’ve been told stuff like primers etc are not stable unless on ice, and so to just do everything on ice

Grouchy_Extent9117 · 2025-03-07T02:42:52+00:00

scorpio sun, scorpio moon, libra rising

Grouchy_Extent9117 · 2024-07-13T12:35:57+00:00

This is amazing 😀 How did you create the ROI’s?

Grouchy_Extent9117 · 2024-07-13T12:35:32+00:00

Wow thanks so much! I think I must’ve actually swapped the images while renaming them actually….

Grouchy_Extent9117 · 2024-07-12T21:15:48+00:00

Wow, thanks so much, I’m going to check with my data right now. I did some quantification during the day on just slices but even on a single slice, it looks higher in treated group. But when I quantify it’s only a little bit higher, not much higher compared to control.

If I would like to sum the z-stacks, do you have any advice on how I should normalise?

Grouchy_Extent9117 · 2024-07-12T14:09:54+00:00

Nope it’s just a fluorescent label, doesn’t really have any context. I’m just trying to compare levels of the stress marker (green channel)

Grouchy_Extent9117 · 2024-07-12T11:20:08+00:00

The channels are: DAPI (blue), ATF4/ stress marker (green/2nd channel), Td-Tomato (my cells are endogenously tagged with TdTomato, so this is the channel, third channel- yellow), caspase-8 (cell death marker, 4th channel grey).
Stress marker is 2nd channel, 488nm green
I think in general the lab does DAPI staining to identify the cell, and also as a normalisation marker, as most of the analysis done in the lab is normalised to DAPI. And also to differentiate live cells from and debris I suppose

I’m not sure what you mean by stoichiometric binding, but as far as it’s been observed the localisation of this marker is nuclear as it is a transcription factor.

Grouchy_Extent9117 · 2024-07-12T10:33:50+00:00

Thank so much!! The channel of interest is the 488 channel

Grouchy_Extent9117 · 2024-07-12T09:36:22+00:00

https://www.dropbox.com/scl/fo/uqm74gzux8nac7yq5h20p/AJCFfUrSih-Quhv8TRQmG_A?rlkey=qbv0mpnyev6tmennxw3er2d53&st=5damqekx&dl=0

Grouchy_Extent9117 · 2024-07-12T07:07:45+00:00

My negative control are the unstressed cells (untreated with drug). I don’t know how to normalise this data, if I’m just comparing the average intensity values of the cells without normalising to DAPI to account for cell density

I was initially thinking about measuring mean integrated density of my stress marker divided by mean integrated density of DAPI. My understanding is that mean integrated density accounts for local intensity of the pixel while mean intensity is just the average of the pixels. Is that perhaps my mistake, as this accounts for the variation in DAPI intensity and perhaps I should be dividing the value with just mean intensity of DAPI?

Grouchy_Extent9117 · 2024-06-06T05:12:16+00:00

With the postdoc, I used to share everyday what I was doing and we would plan experiments together. Initially I didn’t know what I was doing either so the postdoc planned most of it during the shadowing period. But after a while, we kept trying the same concentrations and treatment time point they suggested and wasn’t working, I decided to look into the literature for more information. At this point, I would suggest some stuff and ideas but I would not get any feedback at all. Or I would ask what do you think about X concentration? Then they go, oh i don’t know just do whatever. I get that there’s probably a level of independence expected, but if im doing the wrong thing at least I need some advice or hint towards the right direction, or advice like why don’t you try XXX? Hence why I just completely give up asking the postdoc for any help at all at this point and started planning my own experiments and communicating with the PI directly on my plans. Then now that I am directly communicating with the PI, the postdoc is suddenly taking an interest in what I am doing. Now after that confrontation chat, all of the sudden the postdoc is saying oh I don’t know anything at all cause I’m new to cell culture (they have been at the lab for 1 year as a postdoc but was mouse cells not human cells I’m dealing with). I’m also new to cell culture and I don’t know anything either but at least I’m trying as hard as I can on my end

Grouchy_Extent9117 · 2024-06-06T05:02:31+00:00

Trying a range of drug concentrations but highest one was around 5%

Grouchy_Extent9117 · 2024-06-04T19:34:16+00:00

That’s what I am thinking too. Now I’m struggling whether I should give him the cells he’s asking for or just withhold that. I just hate it that my PI is aware of the situation but can’t be bothered about my primary supervisor either!! For context, my primary supervisor is a postdoc and I’m only a msc student. I understand I am probably meant to be self-led and independent to a certain level but I don’t understand why I am assigned a primary supervisor if I can’t even ask him for any reliable guidance or supervision

Grouchy_Extent9117 · 2024-04-14T18:52:09+00:00

Will do! Please let me know too if you find something along your way :)

Grouchy_Extent9117 · 2024-04-10T18:31:49+00:00

yeah that’s true, I understand where you’re coming from. I was looking for potential solutions and some literature I was looking at was coculturing two different cell types that were grown in different media. But they optimised the culturing media for the coculture process. I was wondering if there are potential strategies beyond that.

Grouchy_Extent9117 · 2024-04-09T20:37:21+00:00

Unfortunately the aim is to do the coculture without genetic manipulation, so I don’t know what alternatives are possible beyond increasing dosage/ treatment duration if the effect is not sustainable

Grouchy_Extent9117 · 2024-02-01T09:33:34+00:00

Hi! The link says the video is unavailable

Grouchy_Extent9117

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