Facs flow cytometer c6

Single cells were prepared as previously described (Henry et al., 2009 (link); Xin et al., 2018 (link)) with minor modifications. Briefly, the ipsilateral cortex was quickly removed and immersed into PBS containing 0.2% BSA (w/v) on ice. The cortices of each group were gently ground on the 70 μm cell strainer at 4°C. Then, homogenates were filtered by passing through a 70 μm cell strainer. The cell suspension was centrifuged at 400 × g for 10 min to obtain precipitation, and the cells were re-suspended with 40% Percoll solution, followed by the addition of 75% Percoll solution at the bottom, and centrifuged at 500 × g for 20 min. Cells were collected from two density gradient interfaces and incubated by these antibodies for 30 min at 4°C: anti-mouse CD45-APC (10311, Biolegend) or anti-mouse CD11b-FITC (101206, Biolegend), to evaluate the populations of infiltrating monocytes/neutrophils (CD11b⁺/CD45^high cells). Flow cytometric analysis was performed with use of FACS flow cytometer C6 (BD Biosciences). CD11b⁺ cell populations were further gated for CD45 expression and divided into a low (microglia) and high population (infiltrating monocytes/neutrophils).

Mice were euthanized and perfused with cold PBS. As described previously [2 (link)], the brain tissue was dissociated mechanically and digested in 6 ml of DMEM medium with 0.6 mg/mL collagenase IV (Worthington) for 30 min at 37 °C. Then, the suspension was filtered with a 70-μm cell strainer (BD Biosciences), and the cells were resuspended in 70% Percoll. Then, the cell suspension was separated from the myelin and debris after centrifugation (700 g, 4 °C, 40 min) on a 37–70% Percoll gradient. The inflammatory cells were carefully collected from the interface of the gradient and washed with 10 ml of PBS. Single-cell samples were incubated with CD45-percp (1:200, BD PharMingen), CD11b-FITC (1:200, BD PharMingen), CD16/32-PE (1:200, BDPharMingen), and CD206-APC (1:200, BD PharMingen) at 4 °C in the dark for 25 min. CD45⁺CD11b⁺ cells were considered as microglia (CD45^+low CD11b⁺) and macrophages (CD45^+high CD11b⁺). CD16/32⁺ and CD206⁺cells were acknowledged as M1 and M2 phenotype microglia, respectively. The subsequent flow cytometric analysis was performed using a FACS flow cytometer C6 (BD Biosciences), and the results were analyzed using FlowJo version 7.6.1.

Apoptosis of PC12 cells was assessed with use of the annexin V-FITC/PI Double Labeling Apoptosis Detection Kit as previously described. The percent of annexin V-positive cells determined over 10,000 acquired events was analyzed with use of a FACS flow cytometer C6 (BD Biosciences, San Jose, CA, USA). All assays were performed in triplicate, and each experiment was repeated three times.

The flow cytometric analysis was employed to detect the apoptosis of HBMECs. In short, the HBMECs were collected by trypsin digestion and rinsed twice with PBS. The suspension was blended with Annexin V‐APC (5 μL) and then added PI (5 μL) to reincubated for 10 min. Finally, the mixture was instantly analyzed by a FACS flow cytometer C6 (FCM; FACSCanto II; BD Biosciences) and the results were analyzed by FlowJo v10.7.1 (Tree Star).