Lack of correlation of SP and CD138⁻ or CD138^low+ subpopulations in MM cell lines. (A) ImageStream analysis of sorted viable DRAQ5⁻ SP and MP cell fractions from OPM1 cell line shows expression of the CD138 antigen. Imaging analysis results are depicted in the following channels: side scatter (SSC), bright-field image (BF), channel 3 empty, CD138-PE, channel 5 empty, DRAQ5. OPM1 cells were imaged at 40×/0.75 magnification and analyzed by IDEAS analytical software. Representative flow cytometric evaluation of CD138 expression in sorted viable 7-AAD⁻ SP and MP cells: abscissa is fluorescence intensity of CD138, and ordinate is SSC. Dot plots show quantification of CD138 expression gated on SP fraction (left) and quantification of CD138 expression gated on MP cells (right): CD138⁻ population, CD138^low+ population, and CD138⁺ population. (B) Quantification of CD138⁻ and CD138^low+ cell fractions in MM cell lines (U266, NCI-H929, MM.1S, MM.1R, RPMI 8226-S, RPMI-Dox40, RPMI-MR20, RPMI-LR5, OPM1, OPM2, OPM6, delta47, OCIMy5, XG-1, JJN3, KMS-11, KMS-18, and KMS-34. (C) Representative dot plots of SP analysis of OPM1 cells, costained with Hoechst 33342 and CD138-PE mAb. Dot plot depicts Hoechst 33342–stained viable cells gated on CD138⁻, CD138^low+, and CD138⁺ populations; gates represent the population of SP cells. (D) Correlation between SP fraction and CD138⁻ or CD138^low+ phenotype in 17 MM cell lines tested (U266, NCI-H929, MM.1S, MM.1R, RPMI 8226-S, RPMI-Dox40, RPMI-LR5, OPM1, OPM2, OPM6, delta47, OCIMy5, XG-1, JJN3, KMS-11, KMS-18, and KMS-34).