For example ; Do we need to grow them back in culture after we obtain them from the patient, then we extract the RNA and Protein?
Can blood cells ( all types) divide on in vitro? Any idea about the doubling time?
Thank you very much in advance ;)
You need to purify PBMC. And you could try to do Comet technique to see the DNA break. I addition, you could try to do IF to see gammaH2AX foci in the nucleus.
Perhaps, you need to select a cell types using a magnetic sorting (quick and efficient) or using rosette step.
If you need to know just the load of DNA damage that happened to the peripheral blood cells, you can use whole blood or isolated PBMC to check "nuclear anomalies" using CBMN Cyt assay
1) Nature Protocols 2, - 1084 - 1104 (2007) doi:10.1038/nprot.2007.77
Cytokinesis-block micronucleus cytome assay
Michael Fenech
or
2) DNA damage and repair can be checked by Using Comet assay, for a better understanding please check the below link
http://cdn.intechopen.com/pdfs-wm/23171.pdf
and
Yes!!! , blood cells can grow in cultures and mostly u'll be selecting PBL (Peripheral blood lymphocytes). The doubling time is 24 hrs and we prefer 48/72 hrs incubation to get a better cell population
The duration of cell cycle phases varies considerably in different kinds of cells. For a typical rapidly proliferating human cell (eg: PBL) with a total cycle time of 24 hours, the G1 phase might last about 11 hours, S phase about 8 hours, G2 about 4 hours, and M about 1 hour.
http://www.ncbi.nlm.nih.gov/books/NBK9876/
MN assay is a good way to identify genomic damage in blood cells from in vitro. For that you have to perform the MN- assay from lymphocyte culture (http://www.nature.com/jes/journal/v23/n2/abs/jes201291a.html)
Similarly gH2AX is also a good molecular tool to determine the DNA damage.
If you wish to do that immediately, without culture, then comet assay is the best single cell analysis that you can come across. I guess, my previous mates have already mentioned about them.
All the best.
One of the best sample for comet assay, is blood sample. In addition, blood tissue are single-cell and not need homogenize(unlike tissues). therefore, after the extraction is ready to use.
Dear Faisal Alzahrani: Giovannelli et al (2003) have described a simple method to measure DNA strand breaks and oxidative damage separately in polymorphonuclear and mononuclear leukocytes, using whole blood without previous cell isolation .
Mutat Res. 2003 Jul 8;538(1-2):71-80.
Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay.
http://www.ncbi.nlm.nih.gov/pubmed/12834756
- Badges
- Science method