Phytoplankton Cell Counting by Flow Cytometry - incommet

AAC17 9/24/04 03:47 PM Page 1212 Phytoplankton Cell Counting by Flow Cytometrybe reduced by custom labeling of oligonucleotideprobes with fluorochromes such as FITC or CY3(Amann et al. 1995).4.2. Cell LabelingThe cell labeling protocol was designed for the identificationof cells but was not optimized for cell counting.4.2.1. Materials1. Hybridization oven set at 46°C2. Microcentrifuge3. Fixatives: formaldehyde (stock at 10%) (see Section3.2.2) and ethanol4. Hybridization buffer: 0.9 M NaCl, 20 mM TrisHCl (pH 7.8), 0.01 sodium dodecyl sulfate (SDS),0% to 50% formamide. For every 1% increase inthe concentration of formamide, the meltingtemperature (T m ) of the hybrid is reduced by0.7°C. The percentage of formamide must beadapted for each probe to ensure a specific labeling(Amann et al. 1995).5. Wash buffer: 0.028 to 0.9 mM NaCl, 5 mM EDTA,0.01% SDS, 20 mM Tris-HCl, pH 7.5. Theconcentration of NaCl must be adapted for eachprobe so the stringency of the washing buffer isequivalent to the stringency of the hybridizationbuffer.6. Resuspension buffer: PBS4.2.2. Procedure1. Cell fixation and permeabilization: Samples (5 mLof cell suspension at 10 5 cells/mL) should be fixedwith formaldehyde (1% final concentration;glutaraldehyde should not be used because itprevents probe binding) and stored at 4°C for 1hour. Cells are then pelleted (3 minutes, 4000 ¥ g)and resuspended in a cold (-80°C) mixture (70 :30,v/v) of ethanol and PBS (500 mL).2. Hybridization: Cells are then pelleted again in anEppendorf type of tube and resuspended in 20 to100 mL of hybridization buffer. A 20-mL aliquot ofthe cell suspension is then incubated at 46°C for 3hours with the oligonucleotide probe (2.5 ng·mL -1 ).An aliquot without probe incubated in the samecondition can serve as a negative control forautofluorescence. Hybridization is stopped by theaddition of 1 mL of cold PBS at a pH of 9.0.Samples are then stored at 4°C until analysis withFCM.4.3. Analysis of Hybridized Cells withFlow CytometryFirst calibrate the sample flow rate (see Section 3.1.5).Set the discriminator to green fluorescence (if the fluorochromeused is FITC). Set all parameters on logarithmicamplification (Fig. 17.3). Events are recorded inlist mode. The flow rate and the cell concentration mustbe adjusted to avoid coincidence. Typically, we analyzesamples for 1 to 2 minutes at a delivery rate of 25 to50 mL·min -1 and the number of events is kept at lessthan 1,000 per second (by diluting samples that are tooconcentrated).4.4. Limits and TroubleshootingThe protocol used for whole-cell hybridization involvesseveral centrifugation steps that generally lead to theFIGURE 17.3. Flow cytometric analysis of fluorescencesignals for whole-cell hybridization of exponentially growingAnkylochrysis lutea (van der Veer) Billard in Honda and Inouye(Pelagophyceae) and Chlamydomonas sp. (Chlorophyceae) withfluorescein isothiocyanate (FITC)–monolabeled probes. Bothspecies are nanoplanktonic (diameter 7 to 10 mm). For eachspecies, the distribution of green fluorescence intensity per cellis plotted on a three-decade log scale. The intensity of green fluorescenceper cell was measured for cells incubated withoutprobe (green autofluorescence, no probe) and in the presenceof a general eukaryotic probe (Euk1209), a Pelagophyceae-specificprobe (Pela 01), and a Chlorophyta-specific probe (Chlo 02).(Modified from Simon et al. 2000.)