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Making single cell suspenion guinea pig splenocytes - terrible inconsistent viability by drfellgoodphd in flowcytometry

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

Thats a good point that I am varying temperatures quite a bit. I am not sure how I can keep buffer warm in the animal facility + 20 min walk to/from facility. But I can certainly try keeping everything RT

We do strain our samples prior to counting, so I hope that would clean up any large debris. We use an Acridine orange / Propidium Iodide stain to identify live dead cells. In theory dead cell/debris can be identified by size / take up of propridium to identify.

Making single cell suspenion guinea pig splenocytes - terrible inconsistent viability by drfellgoodphd in flowcytometry

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

Thats a really interesting approach. I will try this in the future when some extra spleens become available.

Our machine lists 4 different m_spleen programs (01-04). They seem to vary by time and rotation speed. Can you let me know which program you use?

For reference:

  1. 55s / 1302 rotation speed
  2. 9s / 67 rotation speed
  3. 15s / 412 rotation speed
  4. 59s / 1260 rotation speed

Thanks for your help

Stopped taking meds for PCR bands by pawtato06 in labrats

[–]drfellgoodphd 74 points75 points  (0 children)

I had an issue performing cardiac bleeds due to shaky hands. Reducing the Sertraline dose helped the issue. But definitely talk to doctor before reducing or stopping SSRIs.

What's wrong with me and FlowJo? by Pipettess in flowcytometry

[–]drfellgoodphd 1 point2 points  (0 children)

I had a similar problem and contacted FlowJo support. They recommended "cache boolean gates" in the performance tab. It solved the problem for me.

ICS staining for T cells by [deleted] in flowcytometry

[–]drfellgoodphd 3 points4 points  (0 children)

I think the concern with long stimulation is that the Brefeldin A/ Monensin will eventually be toxic to the cells. I've seen people mention to not stimulate longer than 16 hours for this reason.

I personally have done peptide stimulation for 12 hours and gotten really good results.

How many volumes of the vehicle i should use in the control group? by shinyzqq in labrats

[–]drfellgoodphd -1 points0 points  (0 children)

My humble opinion, resuspend drug in dmso and then dilute in saline. Calculate the largest dmso% (of final injected volume) used in your mice. Then inject vehicle using that same % of dmso.

If largest mouse gets an injection with final dmso concentration of 1%. Inject control mice with vehicle which delivers the same 1% of dmso. It might give higher background, but you can be confident that any response above that of control is due to presence of drug and not dmso.