Flow cytometry employs instrumentation that scans single cells flowing
past excitation sources in a liquid medium.@
The technology can provide rapid, quantitative, multi-parameter analyses
on single living (or dead) cells based on the measurement of visible and
fluorescent light emission.@ Flow
cytometry is a widely used method for characterizing and separating individual
cells.@
Polyploid cells are sometimes generated by protoplast fusion during the
PEG-mediated transformation.@ To check
whether a transformant is a polyploid or not, DNA amount should be measured by
flow cytometry.@ This protocol describes
the estimation of ploidy number using a Beckman coulter EPICS XL cytometer.
1. Add 300
µl of the extraction buffer in a 6 cm-dish.@ Place the dish on ice.
2. Put
protonemata ( <100 µl) or gametophores ( <n = 20) into the
extraction buffer and cut them by chopping with razor blade
3. Incubate the
chopped tissue with the extraction buffer for 20 min on ice.@ During the incubation, prepare the filter
units by inserting a Cell Trix filter into a 1.5ml microtube.@
4. Transfer the
lysate to the prepared filter unit by decanting and filter the lysate.
5. Incubate the
flow-through at 37˚C for 10 min.
6. Add 20 µl
of the PI solution to 1/10 vol. of the flow-through and mix by pipetting.
7. Incubate the
mixture at 37˚C for 10 min or 4˚C overnight in darkness.
8. Measure the
fluorescence of each sample with the flowcytometer.@ The user should consult their local flow cytometry facility for
general instruction on flow cytometry and advice on specific procedures for
collection and analysis of samples. @We usually
use a Beckman coulter EPICS XL cytometer.@
Outline for operating the flow cytometer is described below.@
1) Turn on the EPICS XL power
and wait for more than 30 min.
2) Perform quality control.
3) Use the appropriate template
for acquiring data.
4) Place sample tube on EPICS
XL.@ Select gslow speedh at first and
change the appropriate flow speed.
5) Stop the flow and print
analyzed data.
6) Shut down
Solution
required
i) DABCO
solution
Dissolve 125
mg of DABCO (Sigma D2522) in 10 mL of PBS
ii) 1 mg/ml PI
solution
Dissolve 10
mg of PI in 10 mL of DABCO solution
iii) 10 mg/mL
RnaseA (Dnase-free)
iv) Extraction
buffer
@ 1 )Stock solution
10
mM Tris-HCl pH 8.0
2
mM MgCl2
0.1%
TritonX-100
@ 2) Extraction buffer
Stock
sol.@@@@@ 200 ul
RNase
sol.@@@@@@ 2 ul
E Petri dish (60 mm)
E Sample tube for EPICS
E 20 µm Filter
(CellTrix, PARTEC)
E Forceps
E Razor blade
1. Cut the tissue
roughly.@ Excess fragmentation of the
tissue will result in high background.