TERESA MARTIN,1 AHMED ZAFER,2 DAVID KING,1 LAKSHMI KRISHNAN,2
1 ProteinSimple, Ottawa, ON, Canada,
2 National Research Council, Ottawa, ON, Canada,
ABSTRACT:
Determination of cell concentration in biological samples is commonly accomplished using a hemacytometer. However, when samples contain low total concentrations, or rare subpopulations, the small number of cells counted leads to inaccuracies. This can be of practical importance in applications such as early stage cell proliferation and stem/progenitor cell studies which have highlighted the need to accurately quantify low concentration populations. In this study we addressed the linearity and repeatability of hemacytometry for low cell concentration samples, and investigated the use of flow microscopy as an alternative method. A 1:3 dilution series was performed on four sample types (IC-21 murine macrophage cells, EL4 murine T-lymphoma cells, primary murine spleen cells, and 10µm polystyrene beads), with starting concentrations of approximately 300,000/ml. Counts were obtained in triplicate with each method. R² values for hemacytometry ranged from 0.7705 to 0.9926 at concentrations <16,000/ml, with CV ranging from 28-108%. Linearity and repeatability were poor for the hemacytometer at low concentrations. R² values for flow microscopy ranged from 0.9997 to 1.0 at concentrations <16,000/ml, with CV ranging from 2-7%. Flow microscopy showed a high degree of linearity and repeatability at low and high concentrations. Also, flow microscopy provided benefits including a statistically significant data set for both cell count and size, removal of operator subjectivity, images and morphological attributes of the cell populations as well as digital record-keeping.
PRESENTED AT:
2010 ASCB Annual Meeting, Philadelphia PA, USA
2010 ASCB_NRC_ProteinSimple_Quantification of Low Cell Concentrations with MFI.pdf
2010 ASCB NRC ProteinSimple Quantification