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 PEG-mediated transformation. To check whether a transformant is a polyploid or not, the DNA content should be measured by flow cytometry. This protocol describes the estimation of ploidy level using a Beckman Coulter EPICS XL cytometer.
1. Add 600
µl of the extraction buffer in a 35mm-dish.@ Place the dish on ice.
2. Put a
colony protonema and gametophore colony into the extraction buffer.
Segmentalize the colony into 20 pieces with tweezers on ice. The 1~2 cm
colonies are suited for this step.
3. Incubate
the segmentalized tissue with the extraction buffer for 5-20 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 40 µl
of the PI solution 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 flow cytometer.@ 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.@ An 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 (Sigma P4170) 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.