Multicolor Flow Cytometry for Glial Cell Analysis

Mice (n=7+7 in both CUS and microinjection) were sacrificed with CO₂. Tissues were gently homogenized through 70µm cell strainers (#352350, BD Biosciences) on ice as we described previously (24 (link), 32 (link)). Homogenates were blocked in PBS+10% rat serum for 1h with gentle rotation at 4°C. Fluorescent antibody makers (0.5µl/marker, mostly from Biolegend) diluted in 200µl PBS+1%FBS were added and incubated for 1h at 4°C with light protection. For CUS experiment, Plxnb2-PE (#145903), CD11b-BV421 (#101251), CD45-BV650 (#103151), and Glast-APC (#130-123-555) were used. For microinjection experiment, CD11b-FITC (#101206), CD45-PE/Cy7 (#103114), Glast-PE (#130-118-344, Miltenyi), and MHCII-BV711 (#107643) were used. Washed samples were finally resuspended with FC buffer and filtered through 35μm cell strainers into flow tubes (#08-771-23, BD Biosciences). The acquisition was made with BD LSR Fortessa™ (BD Biosciences). Data were analyzed using Kaluza (Beckman Coulter). Percentages (%) of positively stained cells were calculated.

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Xuan F.L., Yan L., Li Y., Fan F., Deng H., Gou M., Chithanathan K., Heinla I., Yuan L., Seppa K., Zharkovsky A., Kalda A., Hong L.E., Hu G.F., Tan Y, & Tian L. (2022). Glial receptor PLXNB2 regulates schizophrenia-related stress perception via the amygdala. Frontiers in Immunology, 13, 1005067.

Publication 2022

CD11b-FITC CD45-PE/Cy7 Glast-PE flow tubes BD LSR Fortessa Kaluza

Buffer Cd11b Cells Fitc Fluorescent antibody Light Mice Microinjection Serum Tissues

Corresponding Organization :

Other organizations : University of Tartu, Beijing HuiLongGuan Hospital, Peking University, Tufts Medical Center, University of Maryland, Baltimore

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Variable analysis

independent variables

CUS (Chronic Unpredictable Stress)
Microinjection

dependent variables

Percentage of positively stained cells

control variables

Mice (n=7+7 in both CUS and microinjection)
Tissues were gently homogenized through 70µm cell strainers
Homogenates were blocked in PBS+10% rat serum for 1h with gentle rotation at 4°C
Fluorescent antibody makers were diluted in 200µl PBS+1%FBS and incubated for 1h at 4°C with light protection
Washed samples were finally resuspended with FC buffer and filtered through 35μm cell strainers into flow tubes
The acquisition was made with BD LSR Fortessa™ (BD Biosciences)
Data were analyzed using Kaluza (Beckman Coulter)

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