Hello. I'd like to perform a Western Blont with samples obtained from 3D culture in matrigel. I've done experiments with 3D cultures in 96 well plates in order to test drug activity, but now I'm trying to seed cells in order to perform a western blot, but the experiment went wrong. I pre-coat wells with agarose 1%, then I add a mixture of matrigel (1mg/ml) and cells. I usually work at a concentration of 100.000 cells/ml, and matrigel and spheroids are nice. But I increased this concentration in order to get a bigger amount of mass, so I seeded 250.000 cells/ml. Matrigel concentration remained the same, 1mg/ml. Matrigel didn't formed as usual and cells formed an aggregate that remained floating in the medium. I don't know if I sould reduce cell number or increase matrigel concentration. But if someone of you know a better method, please, could you share it with me?
Thank you very much.
Dear Fernando,
we usually use much less cells and also less Matrigel in 3D culture.
1) As you changed the cell number, did you check a higher or lower amount of Matrigel? That could be the trick.
2) Else, although not physiologically, you could spin them down 1000 rpm at 4°C, 10 min to ensure spheroid formation. Make sure, when working at 4°C.
4) For the Western Blot, I can just give you an idea: Maybe don't increase the cell number in spheroids but rather collect more than one spheroid (triplicates to be sure). Collecting the the spheroids with cut 100 µL pipette tips into one 1.5 ml reaction tube and regular RIPA lysis should work. I can recommend a method used to unite about 20 spheroids (here 60 000 cells/ml --> 3000/well) before cell lysis.
You can find the protocol about immunoblotting of 3D co-cultures in the thesis "Molecular mechanisms of YAP signalling in the context of cancer" written by Dr. Bailu Xie. (see attached on page 38; or link: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwj58sLNzfTqAhUR2KQKHSz1BCAQFjABegQIAhAB&url=https%3A%2F%2Fresearchportal.bath.ac.uk%2Ffiles%2F189049557%2FThesisBailuXieFinal.pdf&usg=AOvVaw2nMnzAxNg4PCwG4T3pg4QJ).
I hope to be helpful. Good luck with your project!
Dear Fernando,
we usually use much less cells and also less Matrigel in 3D culture.
1) As you changed the cell number, did you check a higher or lower amount of Matrigel? That could be the trick.
2) Else, although not physiologically, you could spin them down 1000 rpm at 4°C, 10 min to ensure spheroid formation. Make sure, when working at 4°C.
4) For the Western Blot, I can just give you an idea: Maybe don't increase the cell number in spheroids but rather collect more than one spheroid (triplicates to be sure). Collecting the the spheroids with cut 100 µL pipette tips into one 1.5 ml reaction tube and regular RIPA lysis should work. I can recommend a method used to unite about 20 spheroids (here 60 000 cells/ml --> 3000/well) before cell lysis.
You can find the protocol about immunoblotting of 3D co-cultures in the thesis "Molecular mechanisms of YAP signalling in the context of cancer" written by Dr. Bailu Xie. (see attached on page 38; or link: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwj58sLNzfTqAhUR2KQKHSz1BCAQFjABegQIAhAB&url=https%3A%2F%2Fresearchportal.bath.ac.uk%2Ffiles%2F189049557%2FThesisBailuXieFinal.pdf&usg=AOvVaw2nMnzAxNg4PCwG4T3pg4QJ).
I hope to be helpful. Good luck with your project!
Hi Fernando,
I would recommend to stick to the number of 100.000 cells / ml and make sure to have a final matrigel amount of at least 50% to ensure gel formation. For a better protein concentration you should increase the number of collected wells/condition.
For protein lysis I usually collect all spheroids with ice cold PBS into reaction tubes, spin down at 4°C and 130xg, aspirate supernatant, wash again with ice cold PBS and spin again and at the end lyse the pellet in RIPA buffer. Afterwards you can use a sonicator or syringe and needle to ensure a better lysis by disrupting the spheroids.
I hope this helps you,
Birga
Hi, I also recommend keeping the number of 100000 cells/mL and matrigel concentration. Remember that enhancing number of cells of your spheroids would has an impact in spheroid's physiology (oxigen, nutrients gradients, necrotic cores, etc.). Afterwards, collect more spheroids per treatment (we use 10) for westerblot and lyse them with RIPA and syring to ensure well disgregation.
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