Journal: Cell reports

Article Title: Neuraminidase 1 regulates neuropathogenesis by governing the cellular state of microglia via modulation of Trem2 sialylation

doi: 10.1016/j.celrep.2024.115204

Figure Lengend Snippet: (A–F) Gene set enrichment analysis of differentially expressed genes (in ) identified pathways involved in (A) myeloid cell development, (B) innate immune system, (C) inflammatory responses to lipopolysaccharide (LPS), (D) classical complement, (E) NF-κB, and (F) Fcγ receptor-mediated phagocytosis. False discovery rate (FDR) < 0.25; NES, nominal enrichment score. (G–K) The qRT-PCR analyses of gene expression in 2- and 4-month-old WT and Neu1 −/− hippocampi ( n = 3) for (G) C1qa , C3 , C3ar1 , C4b , and C9 ; (H) Ccl3 , Cx3cl1 , and Cxcl12 ; (I) Ifnγ , Il1α , and Il1β ; (J) Il6 , Il10 , and Tnf ; and (K) Cd33 , Cd68 , Trem2 , and Tyrobp . Data represented as medians ± quartiles. n.d., not detected; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also and .

Article Snippet: CD33 (F3S8B) rabbit mAb , Cell Signaling , 29621 RRID: none.

Techniques: Quantitative RT-PCR, Gene Expression

Journal: Cell reports

Article Title: Neuraminidase 1 regulates neuropathogenesis by governing the cellular state of microglia via modulation of Trem2 sialylation

doi: 10.1016/j.celrep.2024.115204

Figure Lengend Snippet:

Article Snippet: CD33 (F3S8B) rabbit mAb , Cell Signaling , 29621 RRID: none.

Techniques: Virus, Recombinant, Labeling, Plasmid Preparation, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Isolation, Microarray, Staining, Software, Lysis

Journal: Scientific reports

Article Title: A novel method for isolation of tumor infiltrating myeloid-derived suppressor cells from human lung tumor tissue.

doi: 10.1038/s41598-025-99877-x

Figure Lengend Snippet: Fig. 2. Comparison of FACS, MACS and Rosette-MACS approaches. (A) Yield expressed as number of cells recovered per number of initial tumor cells. (B) Cell viability of isolated tMDSCs. (C) Duration of each protocol (min). (D) Representative flow cytometry biparametric dot plot graphs showing the gating strategy followed to analyse the purity of isolated tMDSCs. (E) Purity represented as the percentage of CD33 + CD11b + HLA-DRlow/- cells following the three isolation protocols analyzed by flow cytometry. (A–C,E) Values were mean ± SD, ***p < 0.001 determined by one-way ANOVA.

Article Snippet: Scientific Reports | (2025) 15:15175 7| https://doi.org/10.1038/s41598-025-99877-x Reagent or resource Source Identifier Antibodies FITC anti-human CD33 (clone REA775) Miltenyi Biotec Cat #130-111-027 PE anti-human CD11b (clone REA713) Miltenyi Biotec Cat #130-110-553 PE-Cy7 anti-human HLA-DR (clone REA805) Miltenyi Biotec Cat #130-111-791 Biological samples Human non-small cell lung cancer tumor biopsies Hospital La Ribera, Alzira See Table S1 Human non-small cell lung cancer peripheral blood Hospital La Ribera, Alzira See Table S1 Chemicals, peptides, and recombinant proteins DMEM/F-12 medium Gibco Cat #31,331-028 Antibiotic–antimycotic Biowest Cat #L0010 Phosphate-buffered saline (PBS) Biowest Cat #X0515 RosetteSep DM-M density medium STEMCELL Cat #15725 Carboxyfluorescein diacetate succinimidyl ester (CFSE) Life technologies Cat # C34554 RPMI1640 medium Biowest Cat #L0498 Fetal bovine serum (FBS) Gibco Cat #A3160502 Critical commercial assays Tumor dissociation kit Miltenyi Biotec Cat #130-095-929 Anti-HLA-DR Ab-coated magnetic beads Miltenyi Biotec Cat #130-046-101 Anti-CD14 Ab-coated magnetic beads Miltenyi Biotec Cat #130-050-201 RosetteSep HLA myeloid cell enrichment cocktail STEMCELL Cat #15272HLA E.Z.N.A. total RNA kit Omega Bio-tek Cat #R6934-02 PrimeScript RT reagent kit Takara Cat #RR037A SYBR Premix Ex TaqTM DNA Polymerase Takara Cat #RR420W Anti-CD8 Ab-coated magnetic beads Miltenyi Biotec Cat #130-045-201 T Cell TransAct Miltenyi Biotec Cat #130-111-160 Oligonucleotides Primer: ACTB, forward: A C A G A G C C T C G C C T T T G C Condalab N/A Primer: ARG1, forward: C C G T G C C C A G A T T C T G A C T T Condalab N/A Primer: IDO1, forward: A T G A A G A A G T G G G C T T T G C T C T Condalab N/A Primer: PD-L1, forward: C C T A C T G G C A T T T G C T G A A C G Condalab N/A Primer: NOS2, forward: C T G G A T T T G G C T G G T C C C T Condalab N/A Primer: COX2, forward: C T T G C A G T G A G C G T C A G G A G Condalab N/A Primer: VEGFA, forward: T G A G C T T C C T A C A G C A C A A C A A Condalab N/A Primer: S100A8, forward: A T G C C G T C T A C A G G G A T G A C Condalab N/A Primer: S100A9, forward: C A A T A C T C T G T G A A G C T G G G G Condalab N/A Primer: LOX1, forward: A G G T C T T C A G T T T C T T T A C T C T C C Condalab N/A Software and algorithms BD FACSDiva version 8.0.1 BD Biosciences RRID:SCR_001456 Kaluza analysis version 2.1 Beckman Coulter RRID:SCR_016182 Prism version 9.2.0 GraphPad Software RRID:SCR_002798

Techniques: Comparison, Isolation, Flow Cytometry

Journal: Journal of clinical & cellular immunology

Article Title: Hotairm1 Controls S100A9 Protein Phosphorylation in Myeloid-Derived Suppressor Cells during Sepsis

doi:

Figure Lengend Snippet: Hotairm1 binding to S100A9 protein in MDSCs from septic patients. PBMCs were isolated from early and late septic patients and depleted of the HLA-DR + cells using biotin-coupled anti-HLA-DR antibody and anti-biotin microbeads, followed by the positive selection of CD33 + LOX1 + cells with biotin-coupled anti-CD33, anti-CD11b, and anti-LOX1 antibodies. (A) The CD33 + CD11b + LOX1 + HLA-DR- cells were fixed in 0.2% formaldehyde to preserve RNA-protein complexes. The whole cell lysate was incubated with 5 μg of anti-S100A9 or anti-IgG isotype control antibody and then cross-linked to protein G magnetic beads overnight at 4°C. RNA was extracted from the immunoprecipitated protein-RNA complexes by Trizol reagent and used for detecting Hotairm1 by RT-qPCR, using human Hotairm1 primer assay. (B) CD33 + CD11b + LOX1 + HLA-DR- cells from late septic patients were transfected with scramble or Hotairm1 siRNA for 36 h. The cells were treated as described in A , and RT-PCR determined levels of Hotairm1. (C) Whole cell lysate of CD33 + CD11b + LOX1 + HLA-DR-cells were cleared by centrifugation and subjected to immunoprecipitation with S100A9 or IgG isotype control antibody as described in A . The immunoprecipitated protein complexes were assessed by immunoblotting using phospho-S100A9 (Thr113) antibody. The membrane was stripped and reprobed with a phospho-p38 antibody. The results are representative of two experiments. (D) CD33 + CD11b + LOX1 + HLA-DR-cells were transfected with scramble or Hotairm1 siRNA for 36 h, then washed and incubated with 1 μg/ml of bacterial LPS for 12 h. ELISA determined the levels of IL-10 in the culture supernatants. The data are mean ± SD of 5 patients per group.*p < 0.05, vs . early septic or control siRNA; **p < 0.05, vs . Hotairm1 siRNA/early septic; #p < 0.05, vs . control siRNA.

Article Snippet: Next, the remaining cells were subjected to positive selection using biotin-coupled antibodies against CD33 (Cat #MA1-19522; Invitrogen, Waltham, MA), CD11b (Cat #130-113-795), and LOX-1 (Cat #130-122-119; both from Milteny Biotec).

Techniques: Binding Assay, Isolation, Selection, Incubation, Control, Magnetic Beads, Immunoprecipitation, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction, Centrifugation, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay