I want to isolate CTCs from whole blood with FACS. Can I culture the isolated cells?
Of course you can. Cells isolated via FACS have been placed in culture for may years now and no special protocol is required. You do how ever have to keep in mind some issues:
1. you FACS machine must be completely sterile (including the internal tubing, and sort environment)
2. Cells needs growth factors to survive. This means that the collection tube must contain serum containing media. The volume of collection medial required will have to determined experimentally
3. Depending on the cell type you are planning on sorting, you may have to use different nozzle size. FACS machines use 20umm -120 umm nozzles to create the stream that carries the cells. The smaller the nozzle the better the stream and faster the sorting of the cells will go. However smaller nozzles create a high shear force that can damage cells. To avoid this problem you have to run some tests first to see which nozzle size is more appropriate for your cells type (e.g. epithelial cells require 100umm nozzle while hematopoietic cells are healthy using 40umm nozzle).
4. It is useful to dedicate one FACS machine to cell sorting and not analysis to ensure that your machine stays sterile. The tubings need to be cleaned with bleach at least once a week and the sheath fluid must be checked for contamination just as frequently. I would start by adding Pen/Strep to the sorted cells for the first few experiments until you have a handle on sterility of the sorted cells
5. Good luck!
Hi,
I'm not too familiar with CTC cultures, but you can use FASC sorting to isolated the cells you want and then culture them. This is performed like regular FACS, except the sorted cells are collected in a tube (or plate) with culture media.
It is important to keep cells cold, keep sterile and not to have aggregates.
I suggest you consult with you FACS facility people.
Of course you can. Cells isolated via FACS have been placed in culture for may years now and no special protocol is required. You do how ever have to keep in mind some issues:
1. you FACS machine must be completely sterile (including the internal tubing, and sort environment)
2. Cells needs growth factors to survive. This means that the collection tube must contain serum containing media. The volume of collection medial required will have to determined experimentally
3. Depending on the cell type you are planning on sorting, you may have to use different nozzle size. FACS machines use 20umm -120 umm nozzles to create the stream that carries the cells. The smaller the nozzle the better the stream and faster the sorting of the cells will go. However smaller nozzles create a high shear force that can damage cells. To avoid this problem you have to run some tests first to see which nozzle size is more appropriate for your cells type (e.g. epithelial cells require 100umm nozzle while hematopoietic cells are healthy using 40umm nozzle).
4. It is useful to dedicate one FACS machine to cell sorting and not analysis to ensure that your machine stays sterile. The tubings need to be cleaned with bleach at least once a week and the sheath fluid must be checked for contamination just as frequently. I would start by adding Pen/Strep to the sorted cells for the first few experiments until you have a handle on sterility of the sorted cells
5. Good luck!
Yes you can cultured the sorted cells after FACS as Dr. Raouf mention here you have to fallow the sterile protocol and some precaution during isolation by FACS.
I routinely isolate rare cells using BD FACS Aria III for culturing and other purposes. In addition to Afshin's anwer, I replace the BD sheath fluid that presumably contains preservative with autoclaved 1X PBS.
its possible, but your must use FACS ARIA or canto. If your only have Calibur, I thing you can't do it. FACS calibur only for assay and if you collect from Calibur, you may have cells with contaminat.
But, you can use MAC cells for sorting and then culturing.
At times during sorting u lose rare cells. to avoid this You can sort rare cells using 'carrier' cells. These could be any cells that have markers different from the population of your interest. That way you will have a nice cushion and will not miss or lose ur cells. I don't know if this technique would be useful for CTCs but u could give it a try.
Possible, provided your short is sterile. Collect the sort in FCS supplemented medium and wahs 3 times before you put in culture.
Thank you for the information. I have done sorting using GFP and I have cultured them. Even after two days I see that the cells have not taken the shape. Is there any minimum cell count for culturing? I want to do cell junction imaging for the positive cell, is there any better way for transient transfections?
Yes. Important consideration:
1) Aseptic technique. Machine may be washed for 1/2 to 1 hour with 70% EtOH, before sorting.
2) Don't include inhibitors like sodium azide, etc., in buffers; use tissue culture grade reagents.
3) Extend the centrifugation of the sorted material, both in terms of time and permissible g values. This is done to increase pelleting of sorted cells.
4) Start from smallest size tissue culture flasks, for e.g. 6- 12-, or 24 well plate format. Remember, lower the cell count, harder to grow.
5) Consider using conditioned medium, if possible.
6) Consider Feeder cells possibilities.
7) For transient as well as stable transfections/transductions, it may be advisable to check and increase transfection efficiency for a given cell type/cell line using the reporter gene construct, e.g. GFP, luciferase, beta-galactocidase. In addition, a number of reagents or electroporation methods are available to test.
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