Hi, I have been having some problems with SH-SY5Y. I want to differentiate them and do immunofluorescence. However I have had some problems with their growth. They were growing normal (first photo), then when I tried to plaque they would clump together and I couldn't distinguish them (second and third photo). It has already happened to me several times, even now they grow in groups in the flask. At first I thought I was not trypsinizing them well, I use 0.05% trypsin for 1 minute in the incubator, then I used 0.25% for longer and they died. Before I tried to homogenize them less times (3 at most), now I do it at least 10 times, and I don't know if that stresses them and they still grow in clusters. Should I give up? I look forward to your advice. Thank you very much.
P.S.
- Culture medium: DMEM/F12 10% FBS 1% pen/strep
- Change medium 2-3 days
- Plaque: 96 well. I've trying different concentrations cells/well: 2500, 3500, 4500 and 5500. According to this paper: https://www.nature.com/articles/s41598-021-03442-1
Hi Ana Palacios Ochoa
Although I can not distinguish the exact cause for this phenomenon, I will provide you with a protocol we carry out for SH-SY5Y. I think it would work well.
1. Aspirate the old medium.
2. Wash with PBS without Mg+2 and Ca+2.
3. Add 1 ml 0.25% Trypsin/EDTA to a 25 ml-flask (3 ml for 75 ml-flask), incubate for 2 min in the incubator. Observe under microscope, if there is non-detached cells or aggregated cell, knock on the flask.
4. Inactivate trypsin in a washing medium (DMEM) (add double amount of the trypsin volume).
5. Centrifuge at 1700 RPM for 5 min.
6. Discard the supernatant.
7. Resuspend in a complete DMEM contains (15% FBS, 1% L-glutamine, 1% sodium pyruvate and a suitable antibiotic).
PI: After researching, I found a pic in ATCC website of the no-adherent SH-SY5Y and they look like those in your pics.
Hi Ana,
I had (and I still have, sometimes) the same problem with SH-SY5Y. I only can give you hope, since this problem very much dissapeared after spliting the cells a few times. I still don´t know exactly why this happen. I use a few tricks to avoid the clumps. I don´t allow them to stand several days without spliting them, even when they didn´t reach a high confluence. I agree with you that excesive homogeneization might cause stress and induce the clumpsing. When I´m not sure, I homogenize them with a P1000, but not many times (smaller diametre of the tip). We suspect that the particular treatment of the flasks/plates to allow a better attachment of the cells, might have something to do, so you could borrow the flask/plates of some neighbour lab to make a test.
¡Buena Suerte!
Hi Ana, it is normal for SH-SY5Y cells to grow in clumps and in suspension sometimes.
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