Wash buffer

Cytometric bead arrays (CBAs, BD Bioscience, Heidelberg, Germany) allow for the simultaneous measurement of up to 30 species-specific (in our study only human-specific) mixed cytokines in sample volumes as low as 50 µL [20 (link),21 (link)]. The results obtained using cytometric-based multiplex bead arrays are comparable to ELISA results [20 (link),45 (link)], and CBAs have been validated on dual-laser flow cytometry platforms, such as the FACSCanto [46 (link)].
For standard curves, lyophilized, human-specific, standardized recombinant cytokines were diluted to 5000 pg/mL with BD assay diluents (BD Bioscience), and were subsequently further diluted 1:1 to 5 pg/mL. For each concentration on the standard curve, 50 µL of the dialysates obtained from the recovery experiments or the initial cytokine solution were incubated with a mixture comprising six human cytokine-specific capture beads (BD Bioscience) for 1 h at room temperature. This assay was followed by incubation with a mixture of six human cytokine-specific detection reagents (BD Bioscience) for 2 h at room temperature in the dark. The beads were washed with wash buffer (BD Bioscience) and centrifuged at 200× g for 5 min. The bead pellets were resuspended in wash buffer and were measured with a FACSCanto dual-laser flow cytometer (BD Bioscience) at 488 nm and 633 nm.

CD29, CD90, CD105, and CD34 MSC markers were quantified using flow cytometric analysis. Florescent Activated cell sorting (FACS) analysis was used. After brief centrifugation, cells were resuspended in a wash buffer (BD Biosciences, Germany). A total of 300 ml of cell suspension was incubated with antibodies against CD29, CD105, CD34, and CD90 conjugated with Allophycocyanin (APC), Cyanine 5 (CY5), Phycoerythrin (PE), and Fluorescein isothiocyanate (FITC) dyes for 45 min at room temperature. Flow cytometry was performed on a FACS Calibur (BD Biosciences, Germany), and Cell Quest software was used for analysis. Cells are identified by flow cytometry, which was positive for CD29, CD90&CD105, and negative for CD34 (Figure 1).
MSCs cells were harvested and labeled with PKH26 fluorescent linker dye (Elberry et al., 2016 (link)).
The remaining thirty animals were divided into five groups (six rats/group):
Cognitive and behavioral tests were performed twice (at the start of the work and the end of the work before euthanasia during the light phase of the light–dark cycles). Blood samples were collected (from orbital sinuses) to measure the serum fasting blood glucose level and insulin level. After the euthanasia, the brains were extracted for biochemical and histological evaluation of the hippocampus.

The capture beads supplied with the kit for the proteins of interest (CBA Flex set numbers; IL-6, 558276; IL-8, 558277; sVCAM-1, 560427; MCP-1, 558287) were vortexed for 15 seconds and diluted 1:100 in bead diluent to make up the bead cocktail. 25μL of the bead cocktail was added to 25μL of sample/standard in a 96-well plate. The plate was mixed on a shaker at 500rpm for 5 minutes before incubating for 1 hour at room temperature. The detector cocktail was prepared in the same way as the bead cocktail, substituting the capture beads for detector antibodies (as per the BD manufacturer’s protocol) and the bead diluent for detector diluent. 25μL of the detector cocktail was then added to the wells and the plate mixed as before and left to incubate at room temperature for 2 hours. After incubation was complete 200μL of wash buffer (BD Biosciences) was added to each well and the plate centrifuged at 1300rpm for 10 minutes at room temperature. The supernatant was discarded and the beads re-suspended in 50μL of wash buffer before being transferred into a tube for analysis.

Cells were incubated with Human TruStainFcX (BioLegend, USA) for 30 min to block the Fc receptors and subsequently stained with CD45-Pacific Blue, CD14-APC, and CD11c-FITC (BioLegend) for 30 min at 4°C. After washing with PBS three times, the cells were incubated with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C. After washing with wash buffer (BD Biosciences) three times, the cells were incubated with specific antibodies against MNSFβ for 30 min at 4°C. Then, the cells were incubated with Cyanine Cy™5-conjugated secondary antibodies (Jackson, USA) for 30 min at 4°C. Approximately 100,000 cells were detected using FACS (BD, USA), and the data were analyzed with FlowJo V10.2.