[Cytometry] Sorting neurons
Doreen.Milius at ist.ac.at
Wed Apr 24 08:50:38 EDT 2013
At our Flow facility we are sorting neurons from 21 day old mice isolated from cortex as well as hippocampus using our FACS Aria III sorter. For dissociation of the brain we use the commercially available kits from Worthington, which work very well and give a high yield in live cells.
For sorting we use 100 µ nozzle and 20 psi, event rates are very high 4000-5000 events, but we keep the flow rate very low between 1 and 2. In dissociated tissue it is very normal to find very many events that are no cells, in our case only 10 to 12 % of the total events in FSC/SSC plots are actually the cells. We also find a large size distribution due to irregular shapes, but no doublets (the kit works really good).
Also make sure that the sorted cells feel happy, sort them into medium and place them into an incubator asap. For RNA isolation we sort directly into lysis buffer. Don't extend the sort to more than one hour, after one hour we already find substantial cell death in the unsorted cells.
Institute of Science and Technology Austria
Am Campus 1
A-3400 Maria Gugging
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Daqian Sun
Sent: Montag, 22. April 2013 22:09
To: cytometry at lists.purdue.edu, (cytometry at lists.purdue.edu),
Subject: [Cytometry] Sorting neurons
We have a user who want to sort neurons isolated from mouse tissue. We have a few technical questions:
1. There are tons of dead cells and debris in his sample. I wonder of anyone have a good protocol for preparation of neuron cell suspension.
2. Currently we are using AriaII, 100um nozzle and 20psi for his sort. Do we need a larger nozzle and lower pressure?
3. There is a literature which suggest to use low flow rate, 1000-3000 cells per seconds for neuron sorting. I'm assuming that the neurons also do not like high differential pressure, is this right?
4. The FSC-W and SSC-W signal distribution are much wider than other type of cells. The user told me that the neuron he isolated are not round in suspension. Does anyone has experience sorting irregular shaped cells? Can we still use signal width for doublet discrimination?
Dr. Daqian Sun
Senior Scientist, Manager
Mount Sinai School of Medicine
Flow Cytometry Shared Resource Facility New York, NY 10029
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