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The B cells were also PDGFR inhibitor stained for CD40 activation marker, a candidate for contributing to autoimmune processes in which B cell activation may play a role [32]. We assessed the CD40 MFI of MOG-specific B cells in MS and HI. No significant difference was observed between RRMS (n = 12) and HI (n = 11) in the MOG-BBR subset (Supplementary Figure 2e). 3.3.2. Proliferation State Ki-67, expressed during all active phases of the cell cycle [33], was used to assess the proliferative status of MOG-specific B cells in RRMS patients and HI. Although the detection of KI-67 was difficult in unstimulated B cells, a trend (P = 0.06) was found for the two cohorts: 1.44 �� 0.37% MOG-BBR positive for KI-67 in MS patients (n = 16) and 3.49 �� 0.86% in HI (n = 17) (Figure 4). MOG-specific B cells were stained more strongly by KI-67 than B cells in MS and HI (P Lapatinib solubility dmso explanations for the decreased frequency of MOG-BBR in MS patients compared to HI, apoptosis was considered. A combination of annexin V (recognizing phosphatidylserine (PS) on the cell surface) and DAPI was used to detect early (annexin V+, DAPI?) and late (annexin V+, DAPI+) apoptotic cells (Figure 5(a)). Ten patients with RR-MS and 10 HI were tested. Early and late apoptosis markers of MOG-BBR showed no difference in MS and HI (for early apoptosis: 6.56 �� 1.38 and 7.29 �� 1.61%, P = 0.88; for late apoptosis: 16.20 �� 2.75 and 17.14 �� 3.53%, resp., Figure 5(b)). Figure 5 Analysis of apoptosis in MOG-specific B cells. (a) We used annexin V and DAPI to characterize MOG-BBR apoptosis cells in MS (n = 10) and HI (n = 10). We analysed annexin V+ DAPI? (early apoptotic cells) and annexin V+ DAPI+ (late apoptotic cells) ... 3.5. Intrathecal MOG-Specific B Cells Although cell numbers in CSF samples reduced intrathecal study, we then checked if MOG-specific B cells accumulated in the spinal fluid of 8 patients with a clinically isolated syndrome (CIS), in whom there was a clinical need for CSF analysis and Liothyronine Sodium in 8 patients with other neurological disorders, both inflammatory and noninflammatory, as controls. All analyses were performed with CSF immediately processed after harvesting. As mentioned in the M&M, only 58% of the samples contained enough cells to perform the test. There was an average of 525 �� 105 B cells (CD19+DAPI?) in fresh CSF samples (ranging from 50 to 2710 B cells) in the tested samples. An example of the gating strategy is represented in Figure 6(a). Cells in CSF samples were selected according to the SSC and FSC parameters as beads and rosettes which had a small size (FSC) and high SSC.