I'm working on mammospheres suspended in the media. I have no enough count to perform flow cytometry. I'm working on PCR for CSCs marker genes as well as the fluorescent markers. The problem is that I couldn't prepare the mammospheres for the fluorescence screening as they are not attached to the flask. What could be the possible solution and If there is another procedure to prove they are Breast CSCs.
Thanks.
Take a look at the attached method from SciGine which uses fluorescence imaging to screen mammospheres.
http://scigine.com/method.php?ID=298842
Here is my protocol:
1. Transfer mammospheres into an Eppendorf tube using a large-bore tip (or cut away the tip of a blue tip).
2. Spin down at low G (150~200)
3. Remove media, wash with cold PBS twice
4. Resuspend mammospheres in buffered 4% formalin for 30 min at room temp
5. Spin down, discard formalin, neutralize with 1% glycine for 30 min at room temp
6. Spin down, wash with cold PBS twice
(* omit steps 4 and 6 if you are looking for GFP or RFP because formalin can kill fluorescent proteins unless you will use antibody for detection)
6. Embed the mammospheres. Selections of embedding methods include OCT frozen medium (go to Step 7) and paraffin (go to step 8)
7. Cover mammospheres with OCT medium (prevent air bubbles), leave on cold bath of dry ice+isopropanol for OCT to set (5-10 min), and store the OCT block in -80C. (you will need a applier to break the Eppendorf tube to get the OCT block for sectioning)
8. Cover mammospheres with warm (40~45C), liquid 1% gelatin or 0.6% agar. Let it set on ice. Cover the gelatin or agar block with 70% ethanol for fixation. Send for processing and paraffin embedding.
(* I prefer gelatin for block medium. If you will stain ECM, then use agar)
Hello Abdulaziz,
Since sphere number is an issue, I hope you won't like to try the above suggested protocol of cryosectioning and that's also a time taking and very tough job to do. When you have a very low number you can't go for multiple number of washings also as it will result in loss. I encountered the same problem with my spheres which were very limited in number and I just put some innovation and it worked very fine.
Do this experiment at night, when your spheres are ready in suspension, then cut your tip from top with surgical blade and suspend a suitable volume of medium containing neurospheres in single wells of a 24 well adherent culture plate. Observe it under microscope, make sure that atleast 2-4 good spheres are transfered per well. After you have transferred them, incubate the cells overnight in culture incubator. Your spheres will adhere to surface but as it's less time they will not shed cells and might not dislodge or disintegrate. Come in th morning, observe them under microscope, they will be adhered properly, give a washing with PBS and fix immediately in NBF or para-formaldehyde. Now you can use your typical IF protocol for staining.
Spheroids are highly dense structures so antibody might not penetrate properly, so I will suggest to use TBS with 0.1% Triton-x for every washing and antibody incubation and dilutions. It will permeate your spheres well so that ab can reach to every cell properly. Do primary ab incubation overnight that will give you best results.
Hope it will help you, please update whether it worked or not.
Cheers !.
You want to be careful to bring spheres to adherent or partially adherent.
I did noticed that once spheres contacted or adhered to plate, gene expression and protein levels (including several ECM and stem cell markers) changed within 12-24 hours.
Dear Chung,
I think you haven't read my answer properly (I have stressed specially on doing expt at night and quickly fix spheres in morning). That's why I said that he has to be as quick so that spheres just attach and he has to fix them immediately after attachment, it's just a narrow time window of 6-7 hrs. I have done that and got massive expression of my proteins after immunofluorescence staining. It's my personal experience
Cheers!
It's been a while since original question but still, maybe someone benefits from this...
Spheres (live or formalin-fixed, suspended in PBS) can be transferred to the wells of 24-well plate with poly-L-lysine coated coverslips at the bottom. Once the spheres sediment they will attach to the coating. Since attachment is electrostatic dead spheres will do that too. Than, if living spheres were used, they need to be fixed. From that point on proceed with regular immunofluorescence, but you may need to increase detergent conc. and Ab incubation time (maybe even overnight).
However, cryotomy will give you real, not confocal, sections.
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