Isolation of tetC^NEG, tetC^INT, and tetC^HIGH PCs and comparison of the quantity of cytoplasmic Ig. (A) The 3 PC subsets were purified from peripheral blood by FAC sorting. A May-Grunwald-Giemsa staining is shown in the top row; immunofluorescence confocal microscopy for FITC-tetC and IgG are shown in the middle and bottom rows, respectively (original amplification 40×/0.75 NA). (B) Top row: representative experiments showing the flow cytometry dot plots analysis of the intracellular expression of Igγ, Igκ, and Igλ light chains by tetC^NEG, tetC^INT, and tetC^HIGH PCs. Bottom row: Bar histograms summarize the results obtained in different donors. Values are expressed as the MFI change (MFI of positive PCs minus MFI of negative PCs) for IgG and Igκ light-chain expression, and as MFI of positive PCs for Igλ light chain. Results represent the mean plus or minus SEM (n = 6). (C) Quantification of the IgG contained in purified tetC^INT and tetC^HIGH PCs. Whole-cell lysates were prepared in Triton X-100, and the lysed cells were centrifuged to remove the cell debris. The quantity of IgG released was assessed by ELISA. Results are expressed as picograms of IgG per cell obtained in 2 different experiments. (D) Comparison of the quantity of IGγ1 mRNA expressed by blood isolated tetC^INT and tetC^HIGH PCs. Values were normalized using as endogenous calibrators those obtained for tetC^NEG PCs in each experiment, which were taken as 1. Results represent the mean plus or minus SEM (n = 6).