Good afternoon everyone,
I am struggling with splitting and passaging of SH-SY5Y cells. I feel that most of the time, when I check 2 or 3 days later, I have very limited growth and it takes a long time to see any increase in confluency. I used to have problems with contamination, so I thought that was the issue, but over the past several months I have rarely had contamination problems and the cells just simply do not grow quickly. Maybe I am just being impatient? How long does it usually take to reach around 70% confluency after a passaging? Is there any way to get cells to grow in confluency more quickly?
Thank you to those who reply!
Hi,
1. Which medium you use? I always used a 1:1 mixture of Eagle's minimum essential medium with non-essential amino acids and Ham's F12 medium with 10%FBS.
2. How many cells you plate? I prefer to plate around 1-2 ×106 cells in a T25 flask
3. I always used 15%FBS during recovering cells from liq N2.
4. Cells reached confluency within 3-4 days.
5. How much trypsin you use and for how long?
HOPE IT WORKS!! Good Luck
Hi Elizabeth...!!
I have worked with SHSY-5Y for quite a few times.
1. I would suggest you to increase your FBS % to 15.
2. I am using F12 Medium and that works completely fine. Cells reach confluency within 3-4 days.
3. These cells grow both as adherent and floating. So centrifuge and recover the floating cells. I always maintain it in high number of cells in the flask so the growth is quite good.
Good luck for your research. Hope it helps.
For the basics and general cell culture, as well as preparation for efficient differentiation, I recommend:
Feles, S.; Overath, C.; Reichardt, S.; Diegeler, S.; Schmitz, C.; Kronenberg, J.; Baumstark-Khan, C.; Hemmersbach, R.; Hellweg, C.E.; Liemersdorf, C. Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies. Methods Protoc. 2022, 5, 58. https://doi.org/10.3390/mps5040058
You need a high initial cell number (N0) of 7.5 x 104 cells/cm² for a successful culture compared to other cell lines. A coating with poly-D-lysine or similar is not necessary. Use Neuromedium (1:1 mix of DMEM and Hams' F12) as culture medium.
Hello! I had this same problem for quite a while, and it seems like things recently got better for me, for the most part. Like many of the other commenters noted, maybe you aren't plating enough cells? That was my main issue. Also, what helps is using a hemocytometer to count the average number of cells within 10ul of the preparation (after you've aspirated and just before you start plating). Also, there is a mathematical formula that has helped us figure out how many cells to plate and how many plates we can make. If you are using 6-well plates, it seems that aiming for 1 million (1x10^6) cells/ml worked well for me (after a few days I had 90% confluency). If you are using 60cm plates, aiming for 2 million (2x10^6) cells/ml worked best for me.
-How many ul of cells to add calculation:
- take the average of the 4 squares
- for us, that usually comes out to a certain number x10^4 cells/ml. What's worked for us is to convert that to 10^6.
- do this formula (for example, we want to have 2 million cells in our plate): [(2x10^6) x 1000]/your average number of cells from the first step
- this will give you the volume of cells you can add to the plate
-How many plates to make calculation:
- estimate the volume of cells (in ml) that is in your conical tube, and of course, convert that to ul
- divide your current volume of cells by the volume you got from the last part of the calculation
- this will give you how many plates you can make.
I am honestly not an expert, just a Master's student who had the same struggle. But anyways, this worked best for me, and I hope this helps!
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