I am trying to isolate neutrophils from mouse bone marrow with percoll (62%). The goal is to stimualte the isolated neutrophils with LPS and afterwards lyse the cells for RNA analysis. I wonder what is the best temperature to work with during isolation (on ice or room temperature) since I read that neutrophils are not easily responding when put on ice? However working on room temperature comes with a cost of activation during isolation?
I also wonder who is experienced in isolating RNA from these cells?
Hello.
I think it is not possible to see the physiological response of the cells on ice. Rather, there is a risk of cold shock and other artefactual gene expression.
We used to induce macrophages from bone marrow derived monocytes. Induction, of course, required several days of incubation in a CO2 incubator at 37°C for each stimulus.
At that time they were RNA extracted with TRIzol. (We actually used more often a Japanese product called ISOGEN which has almost the same composition as TRIzol)
Once the cells were suspended in TRIzol, they are mostly processed at room temperature.
After adding alcohol, the quality of the RNA can be further improved by processing on a spin column such as Qiagen's RNeasy.
Good Luck！
Hi,
Isolation of neutrophils using percoll should be done at RT for sure. Once you isolate, put the cells in media with FBS if you want to stimulate them with LPS. Within 2-3 hrs, cells get adhered. After you are done with the stimulation, you can use this kit for isolation of RNA (considering these cells are sensitive and low life span):
PicoPure™ RNA Isolation Kit, Catalog number: KIT0204.
Good luck!