Magdalena Mosiolek —BIO-PROTOCOL

Home About Us For Authors Submit Protocol Archive

Vera Schoft Viehmarktgasse 2a
1 protocol

Nina Chumak Gregor Mendel Institute, Austrian Academy of Sciences, Austria
1 protocol

Magdalena Mosiolek

Gregor Mendel Institute, Austrian Academy of Sciences, Austria

1 Author merit
Reviewer merit
Editor merit

1 Protocol published

Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS

Authors: Nina Chumak, Magdalena Mosiolek and Vera Karolina Schoft, date: 11/20/2015, view: 7506, Q&A: 1

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. ...

More >

Dejan Đukić

Gregor Mendel Institute

Thank you for the protocol; I have a question regarding one of the steps:
After washing the pollen out from the inflorescences, you mention that the pollen grains can be frozen and kept at -80C; do you first resuspend the grains in a glycerol-based buffer before freezing or directly freeze the grains?
Wouldn't directly freezing them induce the creation of ice crystals that would damage the nuclei?
Also, what procedure do you use to unthaw the nuclei after they had been frozen?

Thank you,

4/25/2019 12:06:22 AM Reply

Vera Schoft

Viehmarktgasse 2a

Hi Dejan,
if I remember correctly (I haven't used the protocol since 2014), we didn't do anything special to freeze the pollen. You can, of course, quick freeze them (by using liquid nitrogen) and thawing them on ice is key. But pollen is really tough, therefore we didn't have problems. We anyways wanted to crush them after freezing and by doing FACS, we only collected the intact nuclei (which can be verified by microscopy after the sort).
The vegetative nuclei are more fragile than the sperm nuclei due to differences in DNA compaction. We also did ChIP with the sperm nuclei and had a hard time sonicating the nuclei to get the required 500bp fragments for sequencing. I wouldn't worry about the sperm nuclei. If you want to use the vegetative nuclei for a protocol that needs special handling, you will have to develop a freezing/thawing protocol on your own, but you can check the nuclei nicely under the microscope and distinguish sperm and vegetative nuclei by their shape and size.
I hope this helps.
Best,
Vera

2019-04-29 02:52:29