Thursday, June 04, 2009

Microbiological Applications of the Accuri C6 Flow Cytometer® System

Cell Counting

Flow cytometry provides a rapid method to identify or classify cells, although most cytometers cannot directly provide the concentration or absolute count of cells in a sample. Concentration values are generally obtained from flow cytometric data either by combining hemacytometer counts with flow cytometric population data (dual platform counting) or by adding an internal microsphere counting standard to the flow cytometric sample (single platform counting). The Accuri C6 Flow Cytometer System can measure volume, and therefore allows calculation of cell concentrations within CFlow® Plus software statistics tables without the addition of counting beads. This eliminates sources of variability associated with dual platform counting and the expense of counting beads. The pre-optimized detector performance of the C6 also minimizes technician-to-technician and day-to-day variability in cytometer set-up and sample analysis.

Methods

Sample preparation

Jurkat and U937 cells were grown in RPMI media supplemented with 10% fetal bovine serum. One milliliter (1 mL) of cells in homogenous suspension obtained from saturated or low-density cultures of each cell line was transferred into triplicate 1.5 mL microcentrifuge tubes. 7-AAD (eBioscience; 5 µL/sample) was added for live/dead cell discrimination as was 50 µL of CountBright™ Absolute Counting Beads (Invitrogen; 52,500 beads/50 µL) to allow comparison of cell counts obtained on the C6 with and without the addition of counting beads in each sample. Samples containing cells or beads only were included as gating controls.

Flow cytometric counts

Cell and bead events per 50 µL were analyzed using a C6 Flow Cytometer system with CFlow Plus software, performance validated with Spherotech 8-Peak (Accuri cat # QA-100) and Spherotech 6-Peak (Accuri cat# QA-110) Validation Beads. The fluidics were calibrated to measure volume from 1.0 mL samples in 1.5 mL microcentrifuge tubes and accurate volume measurement was verified using Accuri Volumetric Validation Beads (Accuri cat # QA-120) prior to sample analysis.

Hemacytometer counts

10 µL of each homogeneous cell suspension was combined with 10 µL trypan blue (1:2 dilution). Live and dead (stained blue) cell counts were obtained using the central counting area (1 mm2). Four separate counts were performed per cell culture.

Calculation of cell concentration

Without Beads: Cell events in 50 µL (R3) x 20 = cells/mL
With Beads: (Cell events in R3 / Bead events in R4) x (52,500 beads / 1.05 mL) = cells/mL
Hemacytometer: Number of Trypan blue excluding cells in 1mm2 x 2 x 104 = cells/mL

Cell cycle analysis of multiploid Caulobacter crescentus cell populations

Methods

Caulobactor cells were fixed with 70% ethanol, washed and re-suspended in sodium citrate buffer plus RNAse, and incubated at 50°C for 3 hours. SYTOX® Green nucleic acid stain (Invitrogen-Molecular Probes) was added according to the manufacturer’s directions, and allowed to stain cells for 30 minutes before analysis, without washing, on the C6.