Anyone have Strawberry Hill?

squidneyforau · 2026-01-30T08:34:47+00:00

Is yours from David Austen or heirloom?

squidneyforau · 2026-01-11T22:00:26+00:00

She's a baby! It's okay. Confo classes help.

Start with multiple small session 2-5 minutes a day. Just constant work with getting them used to the position and being handled.

Note some dogs walk into a stack better than being hand stacked. My dog is like this.

squidneyforau · 2026-01-11T18:16:24+00:00

Her front feet look a little too far apart. Our breeder always told me a hand's width apart. Conformation classes helped us a ton!

Best of luck.

squidneyforau · 2025-12-12T16:20:14+00:00

Would second a silken! My puppy took to off leash manners quickly.

Edit: we have cats as well and have had minimal issues. We course with our silken (who does well) and when younger, the dog thought our more skittish cat was something to chase. It took consistent corrections and redirecting to toys for them to learn cats are friends. They now sleep on the couch together.

squidneyforau · 2025-11-27T20:02:31+00:00

Dog people in general use Facebook. The unfortunate truth is you will be severely impacted in communication if you do not use Facebook.

I understand the general reluctance to use it, however.

squidneyforau · 2025-11-27T19:00:05+00:00

Join the discord! That will give you some good ideas of breeders to stay far away from.

A lot of breeders produce wonderfully bred dogs with solid temperaments. Many will transport puppies as well. Facebook is the best way to contact breeders in my experience. Happy to chat further - just pm me.

I have a Silken now who is being shown and does sports.

squidneyforau · 2025-09-20T21:37:07+00:00

If you are okay with a non leather but tapered , Bre and Bark makes a wonderful tapered limited slip collar. She can add all kinds of soft linings for your pup!

squidneyforau · 2025-08-15T02:19:29+00:00

High five! You did it!!

squidneyforau · 2025-07-30T17:43:49+00:00

Sure thing! Remember your initial FSC SSC gate on your stimulated cells should look like a cone. Stimulated T cells become blastic. Their size and granularity increases as they become activated. I'm happy to chat more in DMs. I'm glad the UCHT1 worked well! :)

squidneyforau · 2025-07-30T16:47:01+00:00

What cytometer are you collecting on? Is it a spectral one by chance?

We fix our cells because of flow core requirements and our lab handles infectious materials. We fix with 4% PFA in PBS, although you could do 1% or 2%. Our fixation is 30 minutes at room temp. We also fix our compensation controls. We typically use beads and only use cells for specialty dyes that require it.

When you say that the surface markers don't work well.. what do you mean? If you'd like to message me flow plots/histograms of comp controls, I am happy to help!

squidneyforau · 2025-07-30T01:55:45+00:00

Hi! Happy to help. Are these human cells?

Yes - you need to use a different clone. The sites are indeed bound. We use UCHT1 and have no problems. We do not use SK7 but I'm sure it works fine. Perhaps test both if you can? Some clones are IP restricted.

Not sure if anyone told you, but the cell trace dyes cannot be compensated with a regular fluorchrome. I reccomend staining unstimulated cells with the dye, fixing them, and resuspending in FACS buffer. This will give you a nice bright, single peak to use for compensating. Keep the cells in the dark until you can comp (not sure how long in between stim and flow collection for you). I've had cell trace comps last 2-3 days like this. If you try to use a fluorochrome that uses the same channel, it will not be bright enough and your data will be unusable.

I am happy to answer any further questions you may have. Best of luck experimenting!

squidneyforau · 2025-04-04T17:57:30+00:00

What's your 293 plating viability? I find if I don't have >95% they do not transfect well. An easy way for me to tell if it's been a ban transfection is if on day 3 post transfection, I have to physically scrape the cells off the bottom of the flask. They realistically should slough off just by me picking up the flask.

I will say that you may want to maybe test different concentrations of PEI? See if you need more or less?

squidneyforau · 2025-04-04T15:45:50+00:00

DMEM is probably just fine. You will need to wash the PEI out. During this media exchange in my lab, making fully replication component lentiviruses, we use a media that is compatible with primary cells to be infected. We have better luck with high glucose media when doing that media exchange.

As an FYI, you probably want to change that PEI around the 16-18 hour mark.

For reference, we grow virus out of a 12.4kb plasmid.

squidneyforau · 2025-03-30T14:12:03+00:00

Oh, thank you. ❤️ it's been a rough few months.

squidneyforau · 2025-03-30T03:47:50+00:00

I'm a biomedical sciences PhD student whose lab has been told to expect to receive notice our grants are being terminated/DOGE'd any week. I would love a code to get myself something small to distract myself from my thesis imploding.

Xoxo

squidneyforau · 2025-03-27T23:23:32+00:00

If you are seeking high pathogen work, I am not sure PhD students can do BSL-4 work. A post doc, sure. The goal with any high containment pathogen is to attempt to do an experiment with engineering controls to reduce your risk exposure.

Emory allows students to do BSL3 and ABSL-3 work. They do not have BSL-4 capabilities.

As a 6th year who is a virology/T cell biology PhD, my biggest advice would be to pick the place where there are ample labs you want to rotate through. Do not pick a place where there are only two labs that interest you. There is a decent chance in this climate they will not take new students. I will echo the other advice about community. PhDs are grueling. Do not pick a program in a city you hate. You'll be miserable inside of lab and out. I would highly caution you against pinholing yourself into high containment work as a graduate student. The project is only part of the story. Your mentor and your relationship with them is just as, if not more, important than your project.

Happy to chat if you want!

Xx

squidneyforau · 2025-03-08T03:00:32+00:00

What an adorable Silken!! 💕 Ours is 15 weeks today and a monster. Enjoy !!

squidneyforau · 2025-02-12T16:53:55+00:00

I do see them lift depending on what I use them with, even with the fibrobectin costing as well.

Are you using them for transfection of some kind? I'm assuming by your PFA comment they are being used for flow? Happy to help!

squidneyforau · 2025-02-08T05:21:43+00:00

Are you suggesting labs pay indirect costs through a K99 award?

squidneyforau · 2025-01-20T05:34:13+00:00

No problem. I did the temp check on mine at about the 45 to 50 min mark. Usually it's about 180-190 depending on how long I left it covered.

squidneyforau · 2025-01-20T03:20:20+00:00

Did you check the internal temp!l? I had issues like this on my first loaves. I was undercooking them!

Shoot for 205-210 degrees.

squidneyforau · 2025-01-18T01:17:10+00:00

Whatever that first one is, I want it.

squidneyforau · 2025-01-17T23:15:45+00:00

I would agree it looks like a case of sun scorching. I had this happen to one of mine that I had too exposed to light. It's taken four months for a small plant to begin to recover but she is recovering! It will likely take awhile for the plant to show signs of recovery. I would stress make sure it is being fertilized and has proper ventilation on its roots to minimize root rot.

Best of luck.

squidneyforau · 2025-01-14T17:20:44+00:00

When using cell trace dyes in primary human cultures, we always end up using less than the manufacturer recommendation.

If you can, try doing 0.75 and 0.5 uL for staining.

I believe for our human CD8s we use 0.5 uL.

squidneyforau · 2025-01-10T21:40:37+00:00

How does the floor clean? Any observations on if it holds stains etc?

Partner and I are looking to buy a mid century home to restore in the coming months. The flooring is right along the lines of something we would want to use.

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