My instructions say in one step to "aspirate the supernatant completely". My instructions are below, as well as my question.
Here are the instructions:
1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate
supernatant completely.
3. Resuspend cell pellet in 300 µL of buffer per 10⁸ total cells.
4. Add 100 µL of FcR Blocking Reagent per 10⁸ total cells.
5. Add 100 µL of CD133 MicroBeads per 10⁸ total cells.
6. Mix well and incubate for 30 minutes in the refrigerator
(2−8 °C).
7. (Optional) Add staining antibodies, e.g., 50 µL of CD133/2
(293C3)‑PE (# 130‑090‑853), and incubate for 5 minutes in the
dark in the refrigerator (2−8 °C).
8. Wash cells by adding 1−2 mL of buffer per 10⁸ cells and
centrifuge at 300×g for 10 minutes. Aspirate supernatant
completely.
9. Resuspend up to 10⁸ cells in 500 µL of buffer.
In step 2 and step 8, it says to "aspirate the supernatant completely". Does this mean the supernatant will not be used, or will the layer below the supernatant not be used? Which one will be resuspended in step three and step 9!? In other words, will I aspirate the supernatant and resuspend that, or will I aspirate the supernatant and resuspend what what underneath it?
Thank you!
By the way, I am isolating cd133 stem cells from marrow. The datasheet link is https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001254.ashx
Hi Ellie,
in general, if you want to isolate cells, you will always work with the pellet. The supernatant is of interest if you are looking at cytokines, chemokines or proteins in general.
To your questions, you spin your cells, creating a pellet at the bottom of the tube. The supernatant can probably be discarded unless you do other tests on that with a different protocol.
If a protocol talks about 'resuspending' it always refers to turning something solid into a suspension. So yes, you resuspend the pellet (somewhat like a wash step with the spinning used to get the washing buffer out without losing cells).
The last step refers to a maximum concentration: if you have more than 10^8 cells in your pellet, you will need more than 500µL buffer.
hope this helps you
Erik
Hi Ellie, When is is said for resuspending it means something solid pellet, which pellet down (in your case cell pellet) on centrifuging. But be careful that when you take out liquid supernatant, just to aspirate slowly and do not touch the solid pellet which is cell other wise you will lose the cells little bit in aspirating (taking out) supernatant. So just put the tube infron of your eyes look carefully the solid cell pellet and supernatant and then asirate carefully. That's it. All the best!
Aspirate the supernatant simply means just throw the supernatant and work with pellet.
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