completely different approach we employ: harvest spheres, pellet and embed them in agarose, let solidify and embed the cone in paraffin. Section and get really great pictures/IHC/IF. Use Eosin in the water step before going through the alcohol to find your pellets in the paraffin block easily. Contact me, if you need a more precise protocol.

Hi,

what you can do is transfer spheres on to the coated slide with 10ul-20ul media. keep in the incubators for 24hrs . cells beneath the sphere adhere on to the slide.keeping the sphere intact. i have done ICC staining of primary mibbrain spheres on gelatin coated plates .This works. all the best.

Thanks!! I had only tried leaving them on the slides for up to 2 hours. I'll give it a shot, thanks a lot!

You can also try transfering then into glass bottom plates and leaving them in the incubator overnight then use the plates to do your IHC. I'm assuming you need to use the confocal hence the glass slides. I have used glass bottom plates many times for fluorescent staining and they are brilliant. Also you save on expensive antibodies and such as you don't need that much. I use 96 well plates and you can use 100ul to stain they are great. a bit expensive but worth it.

Instead of IHC, why do not you try flourescent staining?. Easy to perform. Just pellet the cells and then stain them in a 100 microliter staining solution. Following 30 min incubation at dark, get 15-20 microliter out of this solution to make coverslip preparation. Finally, examine them under a flourescence microscope! We did. Succesful!

If you don't want to use cytospin, poly-l-lysine is alternative and quick,you can buy poly-l-lysine commercially and dilute 1:10 using H2O and add 40-300 ul (depend on well size) and leave it for 10 minutes and after that, decant extra liquid on slide and add cells or spheres on slide and leave it for 30-1hr at room temperature and follow the after steps what you want to do. I use this technique for most of non-adherent cells.

In addition, i am not sure if making the spheres adhered and then perform an assay would be a good idea because there may happen some shere-irrelevant activities after adhering them. It may be required to think twice!.

completely different approach we employ: harvest spheres, pellet and embed them in agarose, let solidify and embed the cone in paraffin. Section and get really great pictures/IHC/IF. Use Eosin in the water step before going through the alcohol to find your pellets in the paraffin block easily. Contact me, if you need a more precise protocol.

Hi Jochen, great answer! Thanks. Actually we are interested in your protocol. So, i would appreciate it if you could email it to me on "[email protected]". Thanks so much in advance! Also, lets keep in touch please.

Try full culture medium containing 2% methocel (methyl cellulose). Autoclave 2% methocel in a glass bottle, add 100 mL culture medium, and warm the bottle in an incubator overnight. The culture medium with high viscosity should press down the spheres to the slide and improve attachment. Replace half of methocel medium with regular medium 2 days later.

I grow neurospheres and seeded in 24 well plate with glass pre coated with poly-lysine (overnight) and laminin (2 hours). In 24 hours the spheres are dissolving by migration already. If you want more details, please tell me! Good Luck!

Thanks a lot for all your answers, each one of them helps me to better understand what steps I need to take next. Dr. Maurer, that sounds like a very interesting approach and relatively simple, I'll try it right away.

If you need to analyze the cells exactly as they are positioned in the sphere I would strongly recommend Dr. Maurers protocol. If you try to photograph a sphere that is 100-200 micrometer in diameter you will probably end up with blurry pictures (unless employing two-photon or confocal microscopy).

If you don't care about the sphere architecture I would dissociate the spheres into single cells, seed on poly-ornithine/fibronectin, fix with PFA and then stain.

Sincerely,

Tobias

This sounds like many very interesting and nice protocols, but the question is: what exactly do you want to do. Just examine your spheres, than agarose embedding as recommended is perfect. The methyl cellulose approach sound similar interesting, but here the cells will attach and grow. this can easily be achieved (with neurospheres) by using ECM coatings. Then the spheres do differentiate and you will get nice pictures of neurite outgrowth etc. But, as already mentioned, this changes a lot. Do the spheres have to survive, or are you going to fix them. You can easily include them in either collagen or ECM gel, let it polymerize and fix it immediately. We do have protocols for the staining procedures in the gels. Use either confocal or Keyence microscopy to screen through the sphere. You can easily quantify (if the staining is fine).

Best

KH

Dr. Maurer, your protocol sounds good and easy for me. Could you please send me a copy of your protocol at [email protected].

Thanks,

RJ

Dr. Maurer, I agree that your protocol sounds very good! 

Could you send me a copy of it, please? As Mohammad I´m trying to stain tumorspeheres and it have been difficult to transfer them to slides.

I woul be very greatful if you can send me a copy of your protocol at [email protected]

Thanks!

Hi Dr. Maurer, I just read this post and I am interested in your protocol. I would appreciate it if you could email it to me on [email protected].

Thank you!

Dear Dr. Maurer, I am currently struggling with performing an IF on the sphere culture. I am thankful to have read this post and I hope you would share your protocol with me at [email protected]. Thank you in advance.

Dear Dr Maurer, if you could share your protocol with me at [email protected] , it would be really helpful. Thank you in advance.