cd14 microbeads miltenyi biotec  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: Decidual stromal cells drive CD16 + macrophages towards an immunoregulatory phenotype via extracellular matrix-adhesion molecule interaction during early pregnancy

doi: 10.3389/fimmu.2025.1747323

Figure Lengend Snippet: CD16 + macrophages constitute a high proportion in decidua with an immunoregulatory phenotype. (A) Expression of FCGR3A in immune cells from endometrium (Ctrl, n=3) or decidua (NP, n=3) in UMAP plots of scRNA-seq analysis. And the violin plots is shown in (B) . (C) Levels of CD16 in CD45 + CD14 + macrophages from endometrium (n=10) or decidua (n=10) detected by flow cytometry assays. (D) Expression of CD86, HLA-DR, CD209 and CD206 in CD14 + CD16 + or CD14 + CD16 - dMφ (n=9) in flow cytometry assays. The data are presented as the mean ± SEM; two-tailed Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DMφ from DICs were obtained through positive selection by an CD14 + cell isolation kit according to the reagent instructions (Miltenyi Biotec, 130–050-201).

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Decidual stromal cells drive CD16 + macrophages towards an immunoregulatory phenotype via extracellular matrix-adhesion molecule interaction during early pregnancy

doi: 10.3389/fimmu.2025.1747323

Figure Lengend Snippet: CD16 + macrophages from endometrium and decidua exhibit higher levels of adhesion molecules. (A) Expression of CD49a, CD44 and LYVE1 in CD45 + CD14 + CD16 + or CD45 + CD14 + CD16 - eMφ (n=12) in flow cytometry assays. (B) Expression of CD49a, CD44, LYVE1 and Integrin αVβ3 in CD14 + CD16 + or CD14 + CD16 - dMφ (n=11) in flow cytometry assays. The data are presented as the mean ± SEM; two-tailed Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns.

Article Snippet: DMφ from DICs were obtained through positive selection by an CD14 + cell isolation kit according to the reagent instructions (Miltenyi Biotec, 130–050-201).

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Decidual stromal cells drive CD16 + macrophages towards an immunoregulatory phenotype via extracellular matrix-adhesion molecule interaction during early pregnancy

doi: 10.3389/fimmu.2025.1747323

Figure Lengend Snippet: Patients RM exhibit weakened interactions between DSCs and CD16 + dMφ. (A) Compared the expression of COL4A1 in decidua from women with normal pregnancy (NP, n=8) or recurrent miscarriage (RM, n=8) by Immunohistochemistry. Scale bar, 50 μm. (B) Compared the expression of OPN in decidua from women with NP (n=10) or RM (n=10) by Immunohistochemistry. Scale bar, 50 μm. (C) Compared the expression of HA in decidua from women with NP (n=8) or RM (n=8) by Alcian blue staining assays. Scale bar, 50 μm. (D) Levels of CD16, CD86, CD206 and CD209 in CD45 + CD14 + dMφ from women with NP (n=7) or RM (n=7) by flow cytometry assays. The data are presented as the mean ± SEM; two-tailed Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: DMφ from DICs were obtained through positive selection by an CD14 + cell isolation kit according to the reagent instructions (Miltenyi Biotec, 130–050-201).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A) Immunofluorescence images of live ZsGreen-expressing MCF7 tumorspheres alone or in co-culture with HuMoSC (1:5 ratio) after staining with anti-CD14 (red) anti-Epcam (green) antibodies. Nuclei stained with Hoechst (blue). All images are representative of at least three replicates. White arrows points towards tumorspheres-associated HuMoSC. (B) Tumorsphere formation assay of MCF7 cells cultured alone, with CD52 + HuMoSC or with conditioned medium (CM) from HuMoSC (CM HuMoSC ) or from co-culture of MCF7 and HuMoSC (CM co-culture ). In some conditions, MCF7 were physically separated from HuMoSC cells by a transwell. Statistical significance was assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. ***p<0.001; ns, not significant. Data are represented as mean values ± S.E.M. (C) CellChat inferred outgoing communication patterns of the different cell clusters (left panel) and associated signaling pathways (right panel). (D) Surfaceome workflow for the identification and quantification of total cell surface proteins. (E) Talkien-predicted protein interaction network of HuMoSC and MCF7 surfaceome. (F-G) Kernel density plot showing log2 intensity of TGFB1 in HuMoSC surfaceome (F) and TGF-BR1 (G) in MCF7 surfaceome. CD45 (F) and EPCAM (G) were used as reference for protein intensities. Dashed line: calculated standard deviation (SD) of the three replicates for the corresponding protein. (H) Chord diagram showing predicted TGFb signaling between clusters. Red arrows highlight the signaling specificity of CD52 + HuMoSC towards MCF7_1 and MCF7_2 clusters.

Article Snippet: Monocytes were then isolated from PBMC by magnetic cell sorting using the AutoMACS ® Pro Separator and Human CD14 + cell isolation kit (Miltenyi Biotec) following manufacturer’s indications.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Staining, Tube Formation Assay, Cell Culture, Protein-Protein interactions, Standard Deviation

Journal: Cell Proliferation

Article Title: Abnormal HCK /glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis

doi: 10.1111/cpr.13702

Figure Lengend Snippet: Antibodies for flow cytometry assays.

Article Snippet: The PF cells, isolated from PF of both the control group and patients with EMS, were collected to isolate macrophages with MASC, a human/mouse CD14 + cell isolation kit (130‐050‐201, Miltenyi Biotec, Germany) for in vitro experiments.

Techniques: Flow Cytometry, Fluorescence

Journal: Cell Proliferation

Article Title: Abnormal HCK /glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis

doi: 10.1111/cpr.13702

Figure Lengend Snippet: Deficiency of HCK leads to insufficient phagocytosis of macrophages. (A) The PM cells of patients with or without EMs were incubated with carboxylate‐modified fluorescent latex beads for 1 h (number of macrophages: number of beads = 1:100). Phagocytosis ratio was detected by FCM and calculated as the percentage of CD14 + FITC + cells among CD14 + macrophages. (B) Validation of HCK silenced THP1 cell lines using Western blotting or RT‐PCR. (C, D) HESCs were labelled with PKH26 and co‐cultured with NC ( n = 3) or HCKi ( n = 3) M2 macrophages for 2 h (number of macrophages: number of HESCs = 1:2). Phagocytosis ratio was detected by FCM and calculated as the percentage of PKH26 + macrophages among total macrophages. (E–H) Chicken erythrocytes (CRBC) were labelled with PKH26 and co‐cultured with NC ( n = 3) or HCKi ( n = 3) M2 macrophages (E and G) or PM cells of Hck −/− mice ( n = 4) or WT mice ( n = 4) (F and H) for 1 h (number of macrophages: number of chicken erythrocytes = 1:10). Macrophages labelled with DAPI. Phagocytosis was detected by fluorescence microscopy. The phagocytic index calculated as the percentage of phagocyted chicken erythrocytes among total macrophages (Scale bar, 100 μm). (I–L) The PM cells of Hck −/− mice ( n = 4) or WT mice ( n = 4) were incubated with carboxylate‐modified fluorescent latex beads for 1 h (number of macrophages: number of beads = 1:100). Phagocytosis was detected by fluorescence microscopy (I, J) or FCM (K, L) (Scale bar, 25 μm). Data were presented as mean ± SEM and analysed by t test. * p < 0.05; **p < 0.01; *** p < 0.001. EM, endometriosis; FCM, flow cytometry; HCK, haematopoietic cellular kinase; HCKi, HCK‐silenced; HESC, human endometrial stromal cells line; NC, normal control THP‐1 cells; PM, peritoneal macrophages.

Article Snippet: The PF cells, isolated from PF of both the control group and patients with EMS, were collected to isolate macrophages with MASC, a human/mouse CD14 + cell isolation kit (130‐050‐201, Miltenyi Biotec, Germany) for in vitro experiments.

Techniques: Incubation, Modification, Biomarker Discovery, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Cell Proliferation

Article Title: Abnormal HCK /glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis

doi: 10.1111/cpr.13702

Figure Lengend Snippet: Autophagy restricts macrophage phagocytosis. (A, B) M2 macrophages pre‐treated with regular growth medium or rapamycin (100 nM) for 4 h, and then M2 macrophages were incubated with carboxylate‐modified fluorescent latex beads for 1 h (number of macrophages: number of beads = 1:100). Phagocytosis was detected by FCM and calculated as the percentage of CD14 + FITC + cells among CD14 + macrophages ( n = 6). (C) M2 macrophages pre‐treated with regular growth medium, glutamine‐free medium, rapamycin (100 nM) for 4 h, or bafilomycin a1 (Baf, 100 μM) combine with glutamine‐free medium for 1 h, were incubated with carboxylate‐modified fluorescent latex beads for 1 h (number of macrophages: number of beads = 1:100). Phagocytosis was detected by fluorescence microscopy (Scale bar, 50 μm or 20 μm). (D) M2 macrophages pre‐treated with regular growth medium, glutamine‐free medium, T5224 (10 μM) or T5224 (10 μM) combine with glutamine‐free medium for 4 h, were incubated with carboxylate‐modified fluorescent latex beads for 1 h (number of macrophages: number of beads = 1:100). Phagocytosis was detected by fluorescence microscopy (Scale bar, 50 μm or 20 μm). (E) Schematic diagram of the regulation of glutamine/FOS/JUN‐autophagy‐phagocytosis axis. In the microenvironment of endometriosis, the reduced glutamine level of peritoneal macrophages results in elevated cFOS and cJUN expression. This leads to upregulation of autophagy‐related genes, thereby suppressing macrophage phagocytic function. Restoring macrophage phagocytic ability can be achieved by inhibiting macrophage autophagy (Baf treatment) or by blocking the DNA binding capacity of cFOS/cJUN (T5224 treatment). Data were presented as mean ± SEM and analysed by t test or one‐way ANOVA test. * p < 0.05; ** p < 0.01; *** p < 0.001. NS: no significance.

Article Snippet: The PF cells, isolated from PF of both the control group and patients with EMS, were collected to isolate macrophages with MASC, a human/mouse CD14 + cell isolation kit (130‐050‐201, Miltenyi Biotec, Germany) for in vitro experiments.

Techniques: Incubation, Modification, Fluorescence, Microscopy, Expressing, Blocking Assay, Binding Assay