Fig 1: Validation that primary cultures of the cortex and spinal astrocytes are devoid of B cells.A RT-PCR amplification of Cd20 in rat DI TNC1 astrocyte cell line and rat primary cortex and spinal astrocytes stimulated or not with 200 ng/mL of LPS. cDNA from the spleen served as a positive control. The experiment was also performed on a negative reverse transcriptase sample to rule out genomic DNA contamination. H2O: negative control. A’ RNAseq analyses of Cd20, A” Cd19 or A”’ Gfap in rat primary cortex and spinal cord astrocytes. B Western blot analyses of CD20 in spleen and DI TNC1 cells stimulated or not with 200 ng/mL of LPS during 24 h and 48 h. C Flow cytometry analyses of primary cultures of the cortex and spinal astrocytes with CD45R–CD45RA double labeling and CD45R–CD45RA–GFAP triple labeling. Rat blood served as a positive control. D Comparison of CD markers found in B lymphocytes, plasmocytes and astrocytes (RNAseq data).
Fig 2: Identification of immunoglobulin constant heavy chain in astrocytes.A Immunofluorescence experiments performed on brain (a) and SCI (b) slices using anti-GFAP and anti-IgG. The nuclei were stained with Hoechst. Immunocytochemical labeling of rat primary cortex astrocytes control (A’) or stimulated with 200 ng/mL of LPS (B) using anti-IgG or (C–E) Immunofluorescence with anti-GFAP and anti-IgG performed on rat primary cortex astrocytes control. F Rat primary cortex astrocytes stimulated with 200 ng/mL of LPS during 48 h. (F’) Rat primary spinal astrocytes stimulated with 200 ng/mL of LPS during 48 h. (F’') Magnification of the double staining observed in rat primary spinal astrocytes. G Immunofluorescence with anti-IgG carried out on DI TINC1 cells. H RT-PCR amplification of the complete sequence coding IgG2B constant heavy chain in rat DI TNC1 astrocyte cell line stimulated or not with 200 ng/mL of LPS during 24 h or a mix of cytokines for 24 h and 48 h as well as in rat primary cortex and spinal astrocytes. I Amplification of the mRNA coding the IgG2B transmembrane form. cDNA from the spleen served as a positive control. The experiment was also performed on a negative reverse transcriptase sample to rule out genomic DNA contamination. H2O: negative control. J 5’-RACE-PCR was performed on a cDNA library of DI TNC1 cells or astrocytes from the spinal cord to amplify the whole Igg2B mRNA. Three bands were amplified and sequenced. This revealed that no variable coding sequence was linked to IgG2B coding sequence. However, the presence of a Kozak sequence flanking the 5’-end of IgG2B coding sequence was identified in bands 2 and 3, indicating that this transcript encoded a free IgG2B constant chain. K 5’ Kozak sequence of the IgG2B sequence L Overexpression of rat astrocyte IgG2B transmembrane form in fusion with Flag Tag in DI TNC1 cells. Western blot analysis using an antibody directed against the Flag tag was carried out 24 h, 48 h, or 72 h after transfection. Non-transfected cells and transfection with an empty vector served as negative controls.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for GFAP Antibody (SPM507) [Alexa Fluor® 700]
Available conjugates: Alexa Fluor 700