Journal: iScience

Article Title: Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

doi: 10.1016/j.isci.2023.107626

Figure Lengend Snippet: CCRK is upregulated and selectively expressed in myeloid cells from tumor-bearing mice and patients with HCC (A) Representative flow cytometry dot plots of CD19 + B cells, CD3 + T cells, CD11b + myeloid cells, and NK1.1 + NK cells in splenocytes of naive mice, splenocytes and tumor infiltrating leukocytes (TILs) of RIL-175 tumor-bearing mice. These cells were sorted by FACS and used to detect CCRK expression by western blot. (n ≥ 3). (B) Representative flow cytometry dot plots of CD11b + Gr-1 + MDSCs and CD11b + Gr-1 − myeloid cells from splenocytes and TILs of RIL-175 tumor-bearing mice. CCRK expression was measured by western blot. β-tubulin was served as loading control. Representative data from at least three independent experiments are shown. (n ≥ 3). (C) CD11b + CD33 + myeloid cells sorted from the peripheral blood mononuclear cells (PBMCs) of healthy donors (HD), PBMCs, adjacent non-tumor (NT), and tumor (T) tissues of HCC patients for the measurement of CCRK mRNA expression (relative to GAPDH ) by qRT-PCR. (n ≥ 5). ∗p < 0.05.

Article Snippet: CD33 + MDSCs were then enriched by anti-CD33 mcirobeads (Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: iScience

Article Title: Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

doi: 10.1016/j.isci.2023.107626

Figure Lengend Snippet: Knockdown of CCRK reduces the immunosuppressive activity and induces an immune-stimulatory phenotype of MDSCs (A) Schematic diagram. CD33 + MDSCs were generated from healthy donor PBMCs treated with IL-6/GM-CSF for seven days and purified by CD33 microbeads. Purified MDSCs were transfected with siRNA targeting CCRK (si CCRK ) or control (si Ctrl ). Cells were collected and analyzed by qRT-PCR, western blot, or flow cytometry. The antigen-presenting capacity of MDSCs treated with siRNA was determined by mixed leukocyte reaction (MLR) assay via co-culturing them with CD3 microbeads purified and CFSE-labeled CD3 + T cells from a different donor, at a ratio of 1:1 in the presence of human recombinant IL-2 (20 U/mL). T cell proliferation rate and IFN-γ releasing were analyzed by flow cytometry and ELISA, respectively. (B) CCRK expressions in MDSCs treated with si Ctrl or si CCRK are shown at mRNA (relative to GAPDH ) and protein levels. β-actin was served as loading control. Data are presented as mean ± SD from at least five independent experiments. (C) MDSCs labeled with CFSE before siRNA treatment were used to measure MDSC proliferation and CFSE low cells indicated the proliferating population was analyzed by flow cytometry at day three post transfection. (D–F) (D) The expressions of HLA-DR, (E) proportions of CD68 + HLA-DR + macrophages, as well as (F) CD86 and CD80 surface expressions on CD68 + HLA-DR + macrophages were upregulated upon CCRK knockdown in MDSCs. (G and H) (G) MLR assay showed an up-regulated allogenic T cell proliferation indicated by CFSE low proportion and (H) IFN-γ production in T cells co-cultured with MDSC-si CCRK compared to controls. (n = 7). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S5 .

Article Snippet: CD33 + MDSCs were then enriched by anti-CD33 mcirobeads (Miltenyi Biotec).

Techniques: Knockdown, Activity Assay, Generated, Purification, Transfection, Control, Quantitative RT-PCR, Western Blot, Flow Cytometry, Mlr Assay, Labeling, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: iScience

Article Title: Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

doi: 10.1016/j.isci.2023.107626

Figure Lengend Snippet: CCRK activates STAT3/E4BP4-dependent transcriptional regulation to maintain the IL-10/IL-12 balance in myeloid cells (A) CD33 + MDSCs were generated, purified, and transfected with si Ctrl or si CCRK . The mRNA levels of 13 genes (relative to GAPDH ) determined by qRT-PCR are shown in heatmap. (B and C) (B) IL-10 level, (C) protein expressions of p-STAT3 Tyr705 , STAT3, ARG-I, and E4BP4 were detected in si Ctrl - or si CCRK -treated MDSCs at 48-h by ELISA or western blot, respectively. (D) The expressions of p-STAT3 Tyr705 and ARG-I in si Ctrl - or si CCRK -treated MDSCs were further confirmed by intracellular flow cytometry analysis and are shown in overlay histograms. (E) Western blot analysis of CCRK, p-STAT3 Tyr705 , STAT3 and E4BP4, and qRT-PCR analysis of CCRK , E4BP4 , IL-10 , and IL-12 mRNA levels (relative to GAPDH ) in vector- or CCRK-U937 stable cells. (F and G) (F) qChIP-PCR analyses of STAT3 on E4BP4 and ARGI promoters, and (G) E4BP4 on the promoters of IL-10 and IL-12a in vector- or CCRK-U937 stable cells. The promoter regions for TF binding of indicated genes are shown. (H) Western blot analysis of CCRK and E4BP4, and qRT-PCR analysis of E4BP4 , IL-10 and IL-12 mRNA levels (relative to GAPDH ) in U937-CCRK cells treated by sh Ctrl or sh E4BP4 are shown. β-actin was served as loading control. Data are presented as mean ± SD from at least five independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S6 .

Article Snippet: CD33 + MDSCs were then enriched by anti-CD33 mcirobeads (Miltenyi Biotec).

Techniques: Generated, Purification, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Plasmid Preparation, Binding Assay, Control

Journal: iScience

Article Title: Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis

doi: 10.1016/j.isci.2023.107626

Figure Lengend Snippet:

Article Snippet: CD33 + MDSCs were then enriched by anti-CD33 mcirobeads (Miltenyi Biotec).

Techniques: Staining, Virus, Plasmid Preparation, shRNA, Recombinant, Expressing, Transfection, Magnetic Beads, Protein Extraction, Bicinchoninic Acid Protein Assay, Purification, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Exosomes shed from late sepsis MDSCs inhibit LPS-induced secretion of S100A9 protein from early sepsis MDSCs. Gr1 + CD11b + cells were isolated from bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. Early sepsis Gr1 + CD11b + cells were cultured in 12-well plates with exosomes (50 μg/well) for 24 hr with or without 0.1 μg/ml of E. coli lipopolysaccharide (serotype 0111:B4; Sigma-Aldrich, St. Louis, MO). Levels of S100A9 protein in the culture super natants were measured by ELISA. Data are expressed as means ± SD of 6-8 mice (6-8 cultures/group) from three experiments. * p /** p < 0.05, versus exosomes from sham or early sepsis.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Late sepsis MDSC-derived exosomes switch naïve Gr1 + CD11b + cells into the immnosuppressive phenotype. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were cultured for 24 hr in serum-free media, and exosomes were purified from the culture supernatants using exoEasy Maxi kit. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The CD4 + T cells were cultured with naive Gr1 + CD11b + cells (1:1 ratio) in the presence of exosomes (50 μg/well) for 3 days, and anti-CD3 plus anti-CD28 antibodies (1 μg/ml each) were added to the culture to activate T cells. The cells were harvested, and T cell proliferation was determined by the step- wise dilution of CFSE dye in dividing CD4 + T cells using flow cytometry. (A) Representative dot plots of CFSE positive T cells gated on CD4 are shown. (B) Summary data of flow cytometry. (C) The culture supernatants were used to determine IFNϒ levels by ELISA. Data are expressed as means ± SD of 6-9 mice (6-9 cultures/group) from three experiments. * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Derivative Assay, Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Hotairm1 expression is increased in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. (A) The cells were cultured for 24 hr in serum-free media. Exosomes were purified from the culture supernatants, and exosomal RNA was extracted with exoRNeasy Starter kit. Levels of Hotairm1 were determined by RT-qPCR using RT lncRNAqPCR Assay Primers (Qiagen). Values were normalized to 18S RNA. (B) Total RNA was isolated from Gr1 + CD11b + cells using TRIzol reagent, and levels of Hotaim1 were determined as in A. Values were normalized to GAPDH RNA. (C) Gr1 + CD11b + cells isolated from naïve mice were cultured for 24 hr without or with exosomes (50 μg/well), purified from late sepsis MDSC culture. The cells were harvested, total RNA was extracted and levels of Hotairm1 were determined as in B. PCR was performed in duplicate. Data are presented relative to sham or media control (1-fold). Data in A and B are expressed as means ± SD of 6-9 mice/group from three experiments. Data in C are expressed as means ± SD of 7 cultures from two experiments. * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Expressing, Isolation, Selection, Magnetic Beads, Cell Culture, Purification, Quantitative RT-PCR, Control

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Knockdown of Hotairm1 in late sepsis MDSCs attenuates their immunosuppressive functions. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. (A) Effects of MDSCs on T cell proliferation and activation. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were then co-cultured (1:1 ratio). T cells were stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each). After 3 days, the cells were harvested, and T cell proliferation and IFNϒ production were determined as in . (B and C) Effect of exosomes lacking Hotairm1 on T cells. Late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. (B) Levels of Hotairm1 in exosomal RNA was determined by RT-qPCR. Values were normalized to 18S RNA. (C) Spleen CD4 + T cells were labeled and cultured with naive Gr1 + CD11b + cells (as described in ) in the presence of Hotairm1-lacking exosomes. T cell proliferation and IFNϒ production were measured as in A. Data are expressed as means ± SD of 5-6 mice/group from three experiments. * p < 0.05.KD, knockdown.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Knockdown, Isolation, Selection, Magnetic Beads, Transfection, Activation Assay, Labeling, Cell Culture, Purification, Quantitative RT-PCR

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Hotairm1 binds to S100A9 in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice using anti-Gr1 antibody and magnetic beads. (A) The cells (pooled from 2 mice) were treated with formaldehyde for reversible cross-linking of RNA-protein complexes. Cell lysates were prepared and immunoprecipitated with S100A9 or IgG antibody, and S100A9 levels were determined by western blot. The cross-linked RNA was extracted from the immunoprecipitated complexes using TRIzol reagent, and Hotairm1 levels were determined by standard PCR. (B) Hotairm1 knockdown relocalizes S100A9 in the cytosol. Late sepsis Gr1 + CD11b + cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. Cytoplasmic and nuclear proteins were extracted, and levels of S100A9 were determined by Western blot. (C) Ectopic expression of Hotairm1 in early sepsis Gr1 + CD11b + cells moves S100A9 to the nucleus. The cells were transfected with Hotairm1 plasmid or a control vector for 24 hr. Cell lysates were prepared and immunoprecipitated with S100A9 or IgG antibody, and S100A9 levels were determined by Western blot. RNA was extracted from S100A9 IP and Hotairm1 levels were determined by RT-qPCR. Values were normalized to IgG IP samples and presented relative to vector. (D) Protein extracts were prepared, and levels of S100A9 were determined by Western blot. The results are representative of three experiments.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Isolation, Magnetic Beads, Immunoprecipitation, Western Blot, Knockdown, Transfection, Expressing, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: Overexpression of Hotairm1 in early sepsis Gr1 + CD11b + cells switches them to the immunosuppressive phenotype. Gr1 + CD11b + cells were isolated from the bone marrow of early septic mice using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1 expression plasmid or an empty vector and cultured with 10 ng/ml of recombinant mouse IL-10. (A) After 24 hr, a portion of the cells was used for Hotairm1 expression measurement by RT-qPCR. The remainder of the cells were washed and stimulated with 1 μg/ml of of E. coli lipopolysaccharide (serotype 0111:B4; Sigma) for 24 hr. (B) The cells were harvested, lysed and analyzed for arginase activity. (C and D) Levels of IL-10 and TNFα in the culture supernatants were measured by ELISA. Data are expressed as means ± SD of 5-6 mice (5-6 cultures/group) from two experiments, * p < 0.05.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Over Expression, Isolation, Magnetic Beads, Transfection, Expressing, Plasmid Preparation, Cell Culture, Recombinant, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of clinical & cellular immunology

Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

doi:

Figure Lengend Snippet: High levels of Hotairm1 in plasma exosomes and MDSCs from late septic patients. (A) Exosomes were purified from plasma and RNA was extracted using exoRNeasy Starter kit. (B) Peripheral blood CD33 + CD11b+HLA-DR − cells were isolated by magnetic cell separation. PBMCs were first purified and depleted of the HLA-DR + cells. The HLA-DR − cell population was then subjected to positive selection with biotin-coupled anti-CD33 antibody, followed by anti-CD11b antibody. Total RNA was extracted using TRIzol reagent. Levels of Hotairm1 were determined by RT-qPCR as in . Values in A were normalized to 18S RNA, and values in B were normalized to GAPDH RNA. Data are expressed as means ± SD of 7-9 subjects/group and are presented relative to HC (1-fold), * p < 0.05, versus HC or early septic; ** p < 0.05, versus late septic. HC, helthy control. (C) S100A9 accumulates in cytosol in CD33+CD11b+HLA-DR − cells during late sepsis. Cytoplasmic and nuclear proteins were extracted from CD33 + CD11b + HLA-DR − cells, and levels of S100A9 were determined by Western blot. The results are representative of two Western blots. (D) Hotairm1 binds S100A9 protein during late sepsis. Cell lysates were prepared from CD33 + CD11b + HLA-DR − cells isolated from late septic patients (n=4) and immunoprecipitated with S100A9 or IgG antibody. S100A9 levels were determined by Western blot. RNA was extracted from S100A9 IP, and Hotairm1 levels were determined by RT-qPCR. Values were normalized to the input samples and are presented relative to IgG IP. The results are representative of two immunoprecipitations.

Article Snippet: The CD33 + CD11b + cell subset was then positively selected by incubating with biotin-coupled human anti-CD33 and anti-CD11b antibodies (Miltenyi Biotech) and isolated with anti-biotin magnetic beads.

Techniques: Clinical Proteomics, Purification, Isolation, Magnetic Cell Separation, Selection, Quantitative RT-PCR, Control, Western Blot, Immunoprecipitation

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: 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: FITC anti-human CD33 (clone REA775) , Miltenyi Biotec , Cat #130-111-027.

Techniques: Comparison, Isolation, Flow Cytometry

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:

Article Snippet: FITC anti-human CD33 (clone REA775) , Miltenyi Biotec , Cat #130-111-027.

Techniques: Recombinant, Saline, Magnetic Beads, Software

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: 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

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: 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: Cells were then stained with the following cell surface antibodies: CD33-fluorescein isothiocyanate (clone REA775; Miltenyi Biotec, Bergisch Gladbach, Germany), CD11b-phycoerythrin (clone REA713; Miltenyi Biotec, Bergisch Gladbach, Germany) and HLA-DR-phycoerythrin-Cy7 (clone REA805; Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated at 4 °C for 10 min. For cell sorting, BD FACSAria III SORP cell sorter was utilized with BD FACSDiva software (BD Biosciences).

Techniques: Comparison, Isolation, Flow Cytometry

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:

Article Snippet: Cells were then stained with the following cell surface antibodies: CD33-fluorescein isothiocyanate (clone REA775; Miltenyi Biotec, Bergisch Gladbach, Germany), CD11b-phycoerythrin (clone REA713; Miltenyi Biotec, Bergisch Gladbach, Germany) and HLA-DR-phycoerythrin-Cy7 (clone REA805; Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated at 4 °C for 10 min. For cell sorting, BD FACSAria III SORP cell sorter was utilized with BD FACSDiva software (BD Biosciences).

Techniques: Recombinant, Saline, Magnetic Beads, Software