Journal: STAR protocols

Article Title: Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing.

doi: 10.1016/j.xpro.2022.101337

Figure Lengend Snippet: Figure 2. Gating strategy for FACS-sorting of wound macrophages (A and B) Representative flow cytometry plots at (A) 4 and (B) 14 days post injury. DAPI- single cells were gated (Gates G1-G4) and expression of CD45 was analyzed. CD45+ cells were gated (G5) and analyzed for expression of CD11b and F4/80. Single CD45+CD11b+F4/80+ wound macrophages (G6) were sorted in 384-well plates for RNA sequencing. CD45+CD11b+F4/80+ wound macrophages can further be analyzed for expression of Trem2, CD301b, and MHC II. Index sorting allows merging FACS data with gene expression data at single cell level.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC Rat anti-mouse Trem2 (237920), 100 tests, dilution 1:10 R&D Systems Cat# FAB17291A; RRID: AB_884527 APC-eFluor780 Rat anti-mouse CD11b (M1/70), 0.2 mg/mL, dilution 1:400 Thermo Fisher Scientific Cat# 47-0112-80; RRID: AB_1603195 eFluor450 Rat anti-mouse MHC II (M5/ 114.15.2), 0.2 mg/mL, dilution 1:200 Thermo Fisher Scientific Cat# 48-5321-82; RRID: AB_1272204 FITC Rat anti-mouse CD45 (30-F11), 0.2 mg/mL, dilution 1:200 Thermo Fisher Scientific Cat# 11-0451-82; RRID: AB_465050 PE Rat anti-mouse F4/80 (CI:A3-1), 100 tests, dilution 1:10 Bio-Rad Laboratories Cat# MCA497PE; RRID: 2098196 PerCP/Cy5.5 Rat anti mouse CD301b (URA-1), 0.2 mg/mL, dilution 1:50 BioLegend Cat# 146809; RRID: AB_2563391 Rat anti-mouse CD16/CD32, 0.5 mg/mL, dilution 1:50 Thermo Fisher Scientific Cat# 14-0161-82; RRID: AB_467133 Chemicals, peptides, and recombinant proteins Betaine (5 M) Thermo Fisher Scientific Cat# J77507.AB (Continued on next page) 2 STAR Protocols 3, 101337, June 17, 2022 Continued REAGENT or RESOURCE SOURCE IDENTIFIER dNTP Thermo Fisher Scientific (Life Technologies) Cat# R0192 EDTA (0.5 M, pH 8.0) solution Thermo Fisher Scientific (Ambion) Cat# AM9261 Ethanol Merck Cat# 1009835000 H2O, sterile Fresenius Kabi 40676.00.00 Liberase TM Research Grade Roche Cat# 05401127001 MgCl2 (1 M) Thermo Fisher Scientific (Invitrogen) Cat# AM9530G Murine RNase Inhibitor New England Biolabs Cat# M0314 S NaCl (5 M) Thermo Fisher Scientific (Invitrogen) Cat# AM9759 Poly(ethylene glycol) avg mol wt 8,000 Sigma-Aldrich Cat# P5413-500G Red Blood Cell Lysis Solution Miltenyi Biotec Cat# 130-094-183 TE buffer (13) Thermo Fisher Scientific (Invitrogen) Cat# AM9858 Tris-Buffer pH 8.0 (1 M) VWR (AppliChem) Cat# A4577.1000 Triton-X 100 Merck Cat# T8787-50ML Critical commercial assays AccuBlue Broad Range ds DNA Kit, incl 9 standards VWR/Biotium Cat# 31007 BD CaliBRITE beads BD Biosciences Cat# 340486 ERCC spike-in Thermo Fisher Scientific (Invitrogen/Ambion) Cat# 4456740 HS NGS Fragment Kit (1–6,000 bp) Agilent Cat# NF-474-0500 Illumina DNA Prep, (M) Tagmentation (96 Samples) Illumina Cat# 20018705 Kapa HiFi HotStart Readymix (23) Roche Cat# 7958935001 Magnetic beads (GE; Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic)) GE Healthcare Cat# 65152105050250 SuperScript II Reverse Transcriptase, incl.

Techniques: Cytometry, Expressing, RNA Sequencing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: TREM2 Expression and Amyloid-Beta Phagocytosis in Alzheimer’s Disease

doi: 10.3390/ijms24108626

Figure Lengend Snippet: Expression of TREM2 mRNA in unstimulated (MED) or LPS + Aβ 42 stimulated- PBMC of 13 Alzheimer’s Disease patients (AD) and 12 healthy controls (HC). The Mann Whitney test was used to compare the mRNA or miRNA expression; the Wilcoxon matched-pair test was used to compare results obtained in different cultural conditions. For ddPCR analysis, the QuantaSoft software version 1.7.4.0917 (Bio-Rad, Hercules, CA, USA) was used to quantify mRNA and miRNAs copies. Data are summarized as median and Interquartile (IQR) (25° and 75° percentile. p values of less than 0.05 were considered significant (* p = 0.02; ** p = 0.005).

Article Snippet: Briefly, unstimulated and stimulated monocytes were washed in PBS, and stained with anti-CD14 PC-7 (IgG2a Mouse, clone: RMO52) (Beckman-Coulter, Rome, Italy); and anti-TREM2 alexa flour 488 conjugated (IgG2a Mouse, clone: 237920) (Bio-Techne, Milan, Italy) mAb for 30 min at 4 °C in the dark.

Techniques: Expressing, MANN-WHITNEY, Software

Journal: International Journal of Molecular Sciences

Article Title: TREM2 Expression and Amyloid-Beta Phagocytosis in Alzheimer’s Disease

doi: 10.3390/ijms24108626

Figure Lengend Snippet: TREM2-expressing CD14 + monocytes in 10 Alzheimer’s Disease patients (AD) and 10 healthy controls (HC); results obtained when cells were cultured in medium alone (MED) or upon LPS and Aβ stimulation are shown The Mann Whitney test was used to compare the percentage of TREM2-expressing CD14 + monocyte in AD and HC, and Wilcoxon matched-pair test for comparison before and after LPS stimulation. Data are summarized as median and Interquartile (IQR) (25° and 75° percentile). p values of less than 0.05 were considered significant (* p = 0.05).

Article Snippet: Briefly, unstimulated and stimulated monocytes were washed in PBS, and stained with anti-CD14 PC-7 (IgG2a Mouse, clone: RMO52) (Beckman-Coulter, Rome, Italy); and anti-TREM2 alexa flour 488 conjugated (IgG2a Mouse, clone: 237920) (Bio-Techne, Milan, Italy) mAb for 30 min at 4 °C in the dark.

Techniques: Expressing, Cell Culture, MANN-WHITNEY, Comparison