Hi all,
We are trying to perform a 4C (Circularized Chromosome Conformation Capture) analysis on monocytes isolated from buffy coats.
Just a basic overview of the approach, we perform a standard PBMC isolation, than monocyte isolation by Percoll isolation, and further increase the purity of our monocyte population by MACS selection against CD3, CD19, and CD56. After MACS selection we plate the cells for an hour to let them adhere to the plate, followed by washing with warm PBS, and than we move onto a 4 hour stimulation protocol (with our stimulant/vehicle and warm RPMI). We than fixate our cells with formaldehyde and scrape our cells, and move onto cell lysis as per the 4C protocol.
The trouble we're having is that prior to plating we perform a cell count, but by the time we harvest our cross-linked cells we don't have an idea of how many cells were lost during the adhering stages (especially since buffy coat quality really affects how many monocytes survive the adhering steps). The 4C protocol requires a certain amount of cells in order to run normally (roughly 1*10^7 cells). Counting cross-linked and scraped cells is said to be impossible since the cells are damaged/altered by the cross-linking and scraping, so I'm wondering if there is a convenient way of counting the amount of raw nuclei after cell lysis (nuclei remain intact)?
My main option at the moment is to go in blind, and plate out a huge amount of cells, and perform the nuclei isolation, but I would prefer to have a more precise approach than just performing bulk isolations.
Cheers,
Laszlo Groh
There is absolutely no reason that you can't count nuclei by most of the same methods that you would use to count cells. Disperse them well and count them in a hemacytometer, run them in a FACS etc... It will be easier if you use an aliquot and stain them appropriately for whichever counting method you use.
Hello
Agree withe Prescott Deininger , Find links here may help you
Good luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517210/
https://www.lifetechnologies.com/sa/en/home/references/protocols/proteins-expression-isolation-and-analysis/cell-separation-methods/human-cell-separation-protocols/rapid-quantification-of-human-peripheral.html
The monocyte nuclei can be counted in hemocytometer after dispersion and FACS can also be run.
You can further refer to the following link to get certain valuable informations:-
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC111749/
How was your outcome? I am also using a 4c-seq protocol and I am counting on a haemocytometer staining nucleus previously with trypan blue. It has worked well so far, if you have a more efficient method for this please share :)
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