Gating, sorting, and microscopic examination of 5T2MM cells.

(A) Gating of 5T2MM cells. Cell debris was excluded by a lymphoblastoid gate (R1) on an FSC/SSC dot plot. 5T2MM idiotype (18B9)⁺ cells were defined within a live gate (R2) on an SSC/18B9 dot plot. Autofluoresence was excluded on an 18B9/FL-4 dot plot. No marker was used in the FL-4 channel. 5T2MM cell phenotype was then analyzed on an FL1/FL2 dot plot. Upper panels are dot plots from a bone marrow sample labeled with CD45-FITC, IL-6Rα–PE, and 18B9 with rat-antimouse-PerCP as a secondary reagent. Lower panels are from the same sample stained with isotype-matched control antibody. (B) Sorting and microscopic examination of 5T2MM cells. Dot plots illustrate expression pattern of 5T2MM idiotype (18B9) and CD45 on a bone marrow sample before and after sorting. Photographs below illustrate May-Grünwald-Giemsa–stained cytospin of same sample before and after sorting. These data were obtained from a mouse with 0.57 g/dL paraprotein concentration and 40% of its 5T2MM cells CD45⁺. After sorting, myeloma purity of 97% was obtained, as analyzed by flow cytometry. Microscopic examination of the cytospin indicated a purity of 99%. Original magnification × 400.