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We conducted a binding analysis of rituximab using the viable cells of B-lineage purified from cryopreserved mononuclear cells.
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Ten microlitres from this solution were used to count the viable cells by using a Countess automated cell counter (Invitrogen, UK).
Fold difference in viability between sorted and unsorted populations was calculated using the formula: viable cells in unsorted drug-treated population (% of control)/viable cells in sorted ABCB5+ drug-treated population (% of control).
Alamar Blue dye reduction method was used to measure the viable cells remaining after drug treatment.
Sytox Blue was used to gate the viable cells in combination with CD45, CD19, and CD138 mAbs.
Trypan blue staining was used to count the viable cells, enabling a calculation of the VEGF and IL-6 concentration referred to the number of viable cells.
Cellular viability was examined by counting the viable cells using trypan blue dye exclusion, and cellular proliferation was measured using an MTS proliferation assay kit (Promega, Madison, WI, USA).
Cell proliferation was measured by staining with erythrosin B and counting the viable cells using a hemacytometer.
After incubation for 72 hr, both viable and dead cells were counted by using Guava ViaCount assay, and only the viable cells were included in data analysis.
Acidified ethanol solution was then used to extract the dye from the viable cells and the absorbance of the solubilized dye was then measured [ 48].
Forward and side scatter parameters were used to gate the viable cell population.
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