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Total number of cells was determined by microscopic counting using a hemocytometer.
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We determined the number of pollen grains per spike by calculating the pollen concentration in the wash suspension from microscopic counts using a glass hemacytometer (Hausser Scientific, Horsham, PA).
To verify these results, the effect on cell proliferation was assessed using a MTS tetrazolium assay, and also by microscopic counting of viable (Hoechst 33342 positive/PI negative) cells.
Before use, erythrocytes were quantified by microscopic counting in a hemocytometer; and were then washed with phenol red-free RPMI (lacking fetal bovine serum).
Red blood cell (RBC), hematocrit, and hemoglobin (Hb) concentration were assessed in whole blood EDTA by using an automatic Coulter Counter (Beckman Coulter Inc., CA, USA); reticulocyte (RET) count was performed by microscopic counting on blood smears after vital staining with New Methylene Blue (Reticulocyte Stain, Sigma-Aldrich, St . Louis MO, USA).
Parasitemia was determined by microscopic counting of blood smears or by fluorescence-assisted cell sorting [FACS].
The number of colonies was assessed by microscopic counting.
Urethritis was assessed by microscopic counting of polymorphonuclear leukocytes (PMN) per high power field (HPF); patients with counts of ≥5 PMN/HPF were considered positive for urethritis.
The differential cell proportion was obtained by microscopic counting of 200 cells.
Reticulocyte count was made by microscopic counting on blood smears after vital staining with new methylene blue (reticulocyte stain; Sigma, St . Louis MO, USA).
Roots were significantly colonized by arbuscular mycorrhizal fungi as measured by microscopic counting (33.5% ± 8.4, mean ± SE) and fungal transcript analysis (data not shown).
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