8.　 Flow Cytometry Analysis

Yuji Hiwatashi

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 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.

Procedure

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 “slow speed” 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

Instrument required

・ Petri dish (60 mm)

・ Sample tube for EPICS

・ 20 µm Filter (CellTrix, PARTEC)

・ Forceps

・ Razor blade

Key points to observe