human bone marrow mononuclear cells  (Qiagen)

Journal: bioRxiv

Article Title: IgA displays site- and subclass-specific glycoform differences despite equal glycoenzyme expression

doi: 10.1101/2024.12.11.627887

Figure Lengend Snippet: ( A ) Single cell RNA sequencing analysis UMAP representation of plasma cells sorted from bone marrow of healthy donors. Shown are combined data from 3 donors. Heavy chain subclass information was extracted from VDJ sequencing annotations. Here, only IGHA1/2 are highlighted. Other IGHC isotypes can be seen in Supplementary Fig. 3. ( B ) Dot plot for expression of glycosyltransferases in IgHA1 and IgHA2 expressing plasma cells. Dot colors represent mean expression of the genes in each cell group and dot sizes indicate the percentage of cells expressing the respective genes. ( C ) Schematic overview of the contribution of selected enzymes to glycan processing. MAN1A2 is involved in trimming of the outer-arm mannose residues, B4GALT1 and ST6GAL1 add galactose and terminal sialic acid residues, respectively. ( D ) Flow cytometry analysis of IgA1 and IgA2 producing bone marrow plasma cells. Shown is the mean fluorescence intensity (MFI) for surface 2,6-sialic acid (measured by binding of sambuccus nigra agglutinine) and intracellular MAN1A2 and B4GALT1 (measured by binding of specific antibodies). Data are normalized on the MFI of IgA1 producing plasma cells. Every dot represents one donor. N=5-6. E) ELISpot analysis of IgA1 and IgA2 producing bone marrow plasma cells. Shown is the median and the 75 percentile of the spot size. Significances were tested with Wilcoxon rank test. * – p<0.05; ns – not significant. ( F ) Representative ELISpot images. Scale bar = 2 mm.

Article Snippet: For the single cell sequencing, cryopreserved human bone marrow mononuclear cells from three healthy donors were purchased from STEMCELL TM Technologies (5-50 Million cells per donor; viability > 93 %; two male African-Americans, 30 and 40 years old; one female Caucasian, 36 years old).

Techniques: RNA Sequencing, Clinical Proteomics, Sequencing, Expressing, Glycoproteomics, Flow Cytometry, Fluorescence, Binding Assay, Enzyme-linked Immunospot