cell populations in comBO

[CAMILLA TOMBARI]

Hi TooT,

I saw in the new manuscript that you stained for osteopontin and lipid droplets (for adipocytes) with Bodipy in immunofluorescence. May I ask you at which time point have you performed this readout? Have you tried both at d20 and d35? Moreover, I am wondering how and if you routinely assess the presence of osteo/adipo lineage. If so, do you analyse some markers by flow (es ALP) and/or real time PCR? scRNAseq would definitely be an overkill just for routine QC...

Regarding the population distribution in scRNAseq between d20 and d35 (Suppl. Fig 6), you show changes in the hematopoietic compartment. Have you also observed changes in the stromal compartment, including in the different endothelial types (AEC, CEC and SEC)? 

Also, from the methods section and the legend of Suppl. Fig 5 I don’t understand exactly which samples were frozen and which were fresh before scRNAseq, but I wonder if you have observed differences between the two conditions.

As always, thank you very much!

Camilla

[Abdullah Khan]

Hi Camilla!

Answers to your questions: 

- The images for osteopontin and BODIPY were from d35 I believe 

- We haven't been looking at them too regularly, we usually use ALP in our stromal panel so it's routinely part of our validation assays. I have some time course Osterix, ALP, and FABP4 imaging planned for July! If you're looking for some more regular/thorough validation I would probably do some imaging and flow rather than qRT PCR (too many cell types in there now so not the best routine marker for validation). 

- Yes we see a progressive loss of the endothelial compartment over time which we think is due to the lack of fluid flow. Watch this space.... 

- So for each condition we try and have an n =3/4, and the only way we can really do this is to have a fresh sample and some frozen. Typically we have one fresh sample and we thaw 2x stored vials of frozen material. I did a head to head of the two, and the only major difference is the freeze-thaw reduces your number/quality of EBMs. 

Hope that helps - thanks for tooting and have a lovely weekend, 

Abs

[CAMILLA TOMBARI]

Hi Abs,

your answers are very much appreciated!

May I ask you which protocol do you use to stain BMOs with Bodipy?
1) Do you stain BEFORE or AFTER fixation?
2) In both cases, for how long and at which concentration?
3) Which buffer do you use to dilute Bodipy? Or do you add it straight from your stock solution into the cell culture media?

Thank you and have a nice evening!
Camilla

[Abdullah Khan]

Hey Camilla, 

Bodipy - I just followed the manufacturer's guidelines staining AFTER FIXATION,  for one hour at 1/1000 dilution and that was plenty. It's a reasonably easy dye to use, the background is the only potential problem if you over cook it. 

Abs