Abstract: Objective To prepare stable chicken red blood cells for the calibration of flow cytometry. Methods The traditional
isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2 +-free HBSS treatment and
low-speed centrifugation. The effect of fluroscence staining of the cells was improved by the addition of TritonX-100 to
enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with
the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed
cells. Results Chicken red blood cells obtained by the modified method showed a significantly higher viability than those
obtained by the traditional method [(98.5±3.5)% vs (93.5±2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with
the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the
traditional method [(6.0 ± 0.3)% to 6.2 ± 0.4% vs (8.6 ± 0.5)% to (13.1 ± 1.4)% , P<0.01]. Conclusion The chicken red blood cells
isolated using the modified method can be applicable for calibration of flow cytometry.