I am isolating and culturing the monocytes derived from patient blood samples. The following step followed-
1. Blood + PBS (1:1) mixed and topped up on the histopaque layer. Centrifuged at 400 g, 30, RT.
2. PBMC layer separated and washed with cold PBS by Centrifugation at 400 g, 10 min, 4C.
3. Cell pellets washed again two times with cold PBS by Centrifugation at 300 g, 10 min, 4C.
4. Cells incubated with culture media for 30 min.
5. After incubation, media changed with fresh media and cultured for further growth. (Image Attached)
6. After 24 hrs, media changed with fresh media as per the protocol.
Image taken which shows many small cells other than round monocytes (Attached). What are these cells and how to remove it?
There may be some red cells in there. You can add a 30 second water lysis step. Lyse cells in water and then add an equal volume of 2x pbs. There looks to be some dead cells and debris so you may also want to wash the plate with PBS after the plating step. If you are still having problems you may want to look into a monocyte isolation kit like Miltenyi.
It is hard to tell because I don't have experience with this cell type, but from my experience the small circles in the background look like yeast contamination. Are you using an antibiotic/antimycotic in your media? Does your media turn a strange color (more purple) than normal when there is more of this contamination?
From the second picture, it looks like cell debri, not yeast, as mentiond above. So PBS washing will help (warmed on water bath 37C), between media changes.
It's probably red cells. Try to add 9 ml water then immediately add 1 ml PBS 10x, so your cells would be suspended in PBS 1x. Centrifuge and observe your cells.
Hi Ashok,
First of all - if I understand the protocol correctly you still have lymphocytes and monocytes together. Is this the plan ? Presumably the "round cells" are lymphocytes , which should be in most cells unless you isolate the monocytes in a second step. By a simple staining you can verify which cells you have in front of you. Unfortunately, you have no information about what the target should be.
Greetings
Jochem
Dear Alexis Cecilia Hruby,
Thank you for your comment. It does not happen regularly. Sometimes I received clear monocytes with less small particles as in the image. PBS washing is an option to reduce these but have a chance of cell detachment.
Dear Jochem Spieker,
I am isolating the monocytes from PBMC of the patients blood sample. These images are taken at second stage i.e. after seeding and incubation of the cells for 30 min. As per protocol, only adhered monocyte will be attached to the culture plate after incubation and media change. I will try to confirm the monocyte by CD14 staining.
Dear Ashok,
In addition to the above suggestions, I would like to add that you may require to standardize your culturing protocol.
From your second picture, it seems to me that you may have used a stimulation agent such as PHA/LPA,; you will accordingly need to adjust your culturing density, culturing volume and culturing vessel depending upon the expected/anticipated confluence.
Also sharing a link for thesis from Shodhganga from where I have derived my protocol (please check all centrifuge speeds):
https://shodhganga.inflibnet.ac.in/handle/10603/303166
Regards.,
Rohan Pawar
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