8.@ Flow Cytometry Analysis

Yuji Hiwatashi, Yoshikatsu Sato, and Mitsuyasu Hasebe

 

 

Introduction

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.

 

Procedure

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 speedh 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

 

Instrument required

E Petri dish (60 mm)

E Sample tube for EPICS

E 20 µm Filter (CellTrix, PARTEC)

E Forceps

E Razor blade

 

Key points to observe

1. Cut the tissue roughly.@ Excess fragmentation of the tissue will result in high background.