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. 2023 Oct 25;14:6559. doi: 10.1038/s41467-023-42276-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Experimental flow of Fluorescence-Activated Cell Sorting (FACS)-based microglia (CD45⁺CD11b⁺TMEM119⁺), astrocyte (ACSA-2⁺), and neuron (NeuN⁺) isolation using specific cell markers. b Relative mRNA expression levels (arbitrary units: a.u.) of Cnr1 in microglia, astrocytes, and neurons isolated from wild type mice were measured by quantitative real time PCR (qPCR) using the TaqMan assay protocol. n = 6 mice per group. ***p < 0.001, **p < 0.01 (p values are Microglia versus Astrocytes: p = 0.0010, Microglia versus Neurons: p < 0.0001, Astrocytes versus Neurons: p < 0.0001), determined by one-way ANOVA with post hoc Tukey test. c Relative mRNA expression levels (arbitrary units: a.u.) of Cnr1 in microglia, astrocytes, and neurons isolated from Cx3cr1^CreER/+;Cnr1^+/+ and Cx3cr1^CreER/+;Cnr1^flox/flox mice were measured by qPCR. n = 6 mice per group. ***p < 0.001 (p values are p < 0.0001), determined by unpaired two-tailed Student’s t test. d Microglia-enriched CD11b⁺ cells, ACSA-2⁺ astrocytes, and remaining cells including neurons were collected from the cerebral cortex of Cx3cr1^CreER/+;Cnr1^flox/flox mice and littermate controls (Cx3cr1^CreER/+;Cnr1^+/+) by magnetic activated cell sorting (MACS). For each cell type, expression of Cnr1, marker proteins (Iba1, GFAP, NeuN) and a loading control (GAPDH) in the total protein (arbitrary units: a.u.) were analyzed with SDS-PAGE followed by Western blotting with 10 μg of protein sample loaded in each well. n = 3 mice per group. *p < 0.05 (p values are Cnr1^+/+ Cd11b⁺ cells versus Cnr1^+/+ Astrocytes: p = 0.0495, Cnr1^+/+ Cd11b⁺ cells versus Cnr1^+/+ Remaining cells: p = 0.0204, Cnr1^+/+ Cd11b⁺ cells versus Cnr1^flox/flox Cd11b⁺ cells: p = 0.0479), determined by two-way ANOVA with post hoc Tukey test. b–d Each symbol represents one animal. Data are presented as the mean ± s.e.m.