Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Representative workflow of isolation and culture of adult mouse microglia (A and B) Dissect the brain into small pieces on ice in Petri dish. (C) Collect cell pellets in C-tubes following mechanical/enzymatic dissociation using a gentleMACS dissociator. (D) Preparation of cell straining and debris removal processes. (E) Perform debris removal by carefully overlaying ice-cold PBS onto the cell suspension. (F) After centrifugation, identify three layers; the middle, yellowish layer corresponds to myelin dna debris. (G) Enrich CD11b+ cells by magnetic separation using appropriate columns. (H) Seed isolated microglia onto 6-wll culture plates for downstream assays.

Article Snippet: CD11b Microbead (Clone: M1/70.15.11.5) (1:10 ratio) , Miltenyi Biotech , Cat#130-049-601; RRID: AB_2927911.

Techniques: Isolation, Suspension, Centrifugation

Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Flow cytometry for MACS-isolated microglia purity (A and B) Total CD11b-positive cells from MACS columns; (C) Cell viability assessed by Zombie Red staining; (D) Infiltrating leukocytes, including neutrophils (Ly6G+) and T lymphocytes (CD3+); (E) Proportions of microglia (CD11b+CD45low) versus monocytes/border-associated macrophages (CD11b+CD45high); (F) Proportion of resting (homeostatic) microglia identified as CD11b+TMEM119+ cells.

Article Snippet: CD11b Microbead (Clone: M1/70.15.11.5) (1:10 ratio) , Miltenyi Biotech , Cat#130-049-601; RRID: AB_2927911.

Techniques: Flow Cytometry, Isolation, Staining

Journal: iScience

Article Title: Targeting USP14 enhances immunotherapy response by reprogramming tumor-associated macrophages in colon cancer

doi: 10.1016/j.isci.2026.115362

Figure Lengend Snippet: Targeting USP14 inhibits tumor growth and induces local anti-tumor immunity in vivo (A) Representative images of MC38 tumors harvested on day 21 post-inoculation from mice treated with IU1 (20 mg/kg, i.p., on days 6, 9, 12, and 15) or vehicle control. (B) Statistics of MC38 tumor growth rate following IU1 or vehicle treatment in vivo . The intraperitoneal injection dose of IU1 was 20 mg/kg, administered on days 6, 9, 12, and 15. Data are presented as the mean ± SEM ( n = 6 per group). (C) Spider diagram of the tumor volume growth in each mouse from the IU1 group and the PBS group. (D) Gating strategy for the detection of the TAMs by flow cytometry. We first obtained live cells, and then identified cells that were positive for CD45, CD11b, F4-80, and CD206 as M2 macrophages. (E–P) Proportions of neutrophil (E), M2 macrophage (F), M1 macrophage (G), MDSC (H), activated DCs (I), CD4 T cell (J), Treg cells (K), CD8 T cell (L), IFN-γ + CD8 T cell (M), precursor exhausted T cells (TCF-1 + ) (N), effective CD8 T cells (PD-1 + ) (O) and activated CD8 T cell (CD69 + ) (P) in the TME of the IU1 group and the control group by using flow cytometry. (Q–U) Cytokines IFN-γ (Q), TNF-α (R), IL-2 (S), IL-10 (T), and IL-12 (U) in the TME of each group were detected by Mul-Analyte Flow Assay Kit. (V) Schematic illustration of the proposed mechanism of action of IU1 in reprogramming the tumor microenvironment. Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, and ns: not significant.

Article Snippet: For RNA and protein extraction, CD11b + cells were first enriched using magnetic-activated cell sorting (MACS) with anti-CD11b microbeads (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: In Vivo, Control, Injection, Flow Cytometry