Journal: European journal of immunology

Article Title: Enhanced Mott cell formation linked with IgM Fc receptor (FcμR) deficiency

doi: 10.1002/eji.202250315

Figure Lengend Snippet: In vitro generation of Mott cells and establishment of a Mott hybridoma clone from Fcmr KO mice. (A) Splenic B cells from Fcmr KO (red circles) and WT control (black circles) mice (13–14 wks, male) were cultured in triplicates for 4 days at 37°C in the presence of the indicated stimuli: LPS alone, B-1 stimulation cocktail (LPS/dextran-anti-IgD/IL-4/Il-5), or B-2 stimulation cocktail (anti-CD40/dextran-anti-IgD/IL-4/IL-5). The frequencies of IgM-producing plasmablasts or plasma cells (left), IgM inclusion body-containing cells or Mott cells (middle) and κ + cells among total IgM + cells (right) were assessed by cytoplasmic immunostaining of smears of cultured cells. Results are shown as mean ± 1 SD. Two-sided, independent Student’s t -test was performed for statistical analysis. * P < 0.05, ** P <0.01, *** P< 0.001. (B) Mott hybridoma cells developed from LPS-stimulated splenic B-1 B cells of Fcmr KO mice were cytospun onto glass slides, air-dried, fixed and washed, before staining with PAS reagent (left) and FITC-labeled goat anti-mouse μ antibodies (right). Note cytoplasmic inclusion bodies strongly stained with PAS (left) and FITC-anti-μ antibodies (right).

Article Snippet: After culturing and washing once with PBS/1% FCS, the cultured cells were cytocentrifuged onto glass slides, air-dried, fixed in 95% ethanol/5% glacial acetic acid at −20°C for 20 min, and rehydrated in PBS, before staining with FITC-labeled goat anti-mouse μ and TRITC-labeled goat anti-mouse κ antibodies (both Southern Biotechnol.

Techniques: In Vitro, Control, Cell Culture, Clinical Proteomics, Immunostaining, Staining, Labeling

Journal: European journal of immunology

Article Title: Enhanced Mott cell formation linked with IgM Fc receptor (FcμR) deficiency

doi: 10.1002/eji.202250315

Figure Lengend Snippet: Reversal of the Mott phenotype by introduction of the FcμR. (A) Frequency (%) of cells carrying IgM-inclusion bodies in three cell lines: the original FcμR-deficient Mott hybridoma clone KO-03 (WT), KO-03 hybridoma transduced with empty vector containing GFP cDNA (GFP), and with vector containing both FcμR and GFP cDNAs (FcμR/GFP). Results are shown in mean ± 1 SD from three experiments. Note the significant reduction of cells containing Ig inclusion bodies in the FcμR/GFP transductants. ** P <0.01. (B) ELISA for IgM secretion by the KO-03 Mott hybridoma and its GFP or FcμR/GFP transductants. KO-03 Mott hybridoma (white), GFP-transductants (black) and FcμR/GFP-transductants (red) were plated at 10 5 cells/ml and IgM secreted into the media was assessed by ELISA during the 3-day culture. Results are shown as mean ± 1 SD. From three different experiments. * P <0.05, ** P <0.01, *** P <0.001. (C) Flow cytometric analysis of FcμR expression. An equal number of cell mixture of the original KO-03 hybridoma and their FcμR/GFP (lower) or only GFP (upper) Mott hybridoma transductants was sequentially incubated with MM3 anti-FcμR (right) or isotype matched (IgG1κ) control (left) mAb and then with PE-labeled goat anti-mouse Ig antibodies. (D) Passively acquired IgM on FcμR + /GFP + Mott hybridoma transductant. PE-labeled rat anti-mouse IgM (lower panel) and PE-labeled isotype matched control mAb (upper panel) were used to stain the cell surface of the KO-03 Mott hybridoma transduced with only GFP cDNA (left side) or FcμR/GFP cDNA (right side) and of the original KO-03 hybridoma (WT). The staining profiles of each transductant (clear) were overlayed to that of WT (gray).

Article Snippet: After culturing and washing once with PBS/1% FCS, the cultured cells were cytocentrifuged onto glass slides, air-dried, fixed in 95% ethanol/5% glacial acetic acid at −20°C for 20 min, and rehydrated in PBS, before staining with FITC-labeled goat anti-mouse μ and TRITC-labeled goat anti-mouse κ antibodies (both Southern Biotechnol.

Techniques: Transduction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Control, Labeling, Staining