cd14  (Bio-Rad)

Journal: Frontiers in immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: FIGURE 2 The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A), The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B), The distribution of monocytes of classical (CD14 +CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C), Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D), Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M0, metastasis-negative status, M1, metastasis-positive status. N0, lymph node-negative status, N1-3, lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: FIGURE 6 An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A), Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B), Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C), Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D), Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E), PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F), PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay

Journal: The Scientific World Journal

Article Title: Characterization of the Immune Response Induced by a Commercially Available Inactivated Bluetongue Virus Serotype 1 Vaccine in Sheep

doi: 10.1100/2012/147158

Figure Lengend Snippet: Antibodies used to analyze PBMC populations by flow cytometry.

Article Snippet: Anti-sheep CD14 , Monocytes , IgG 1 , 2 , VMRD , CAM36A.

Techniques: Cytometry

Journal: The Scientific World Journal

Article Title: Characterization of the Immune Response Induced by a Commercially Available Inactivated Bluetongue Virus Serotype 1 Vaccine in Sheep

doi: 10.1100/2012/147158

Figure Lengend Snippet: Mean percentages (± SD) and individual percentages of PBMC populations labeled by antibodies against CD25 + (a, b), B + cells (c, d), and CD14 + (e, f) throughout the experiment. *is statistically significant difference ( P ≤ 0.05) from the mean pre-vaccination value on day 0, based on ANOVA with the Huynh-Feldt correction. Abbreviations: V, day of first vaccination; DFV, day after first vaccination; BV, day of boost vaccination; C, day of challenge.

Article Snippet: Anti-sheep CD14 , Monocytes , IgG 1 , 2 , VMRD , CAM36A.

Techniques: Labeling

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-C: CD14 or CD15 staining was not detected in control human tissue. D-O: CD14 + cells were located both in the plaque of AL ( D , E ) and in the rim of AIL ( G, H ). By contrast, these cells were almost absent in the center or plaque of AIL ( G , H ) and IL ( J, K ). CD15 staining was not detected in any region of the MS lesions ( F , I , L ), although CD15 + granulocytes were clearly detected in the perivascular area of blood vessels ( M - O ). EC, eriochrome cyanine; AL, active lesions; AIL, mixed active/inactive lesions; IL, inactive lesions; Pl, plaque; NAWM, normal appearing white matter. Scale bar: A-C, J-N = 100 µm; D-I = 125 µm; O = 15 µm.

Article Snippet: Slides were washed in TBS before incubation with mouse anti-HLA-DR antibody (1:200; Agilent, M0746, TAL.1B5 clone) and sheep anti-CD14 antibody (1:50; R&D, BAF383) in blocking solution overnight at 4°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-C: CD14 + cells showed different levels of HLA-DR in demyelinating lesions. Based on the intensity of HLA-DR (á′, b′′, ć′) we identified two cell subpopulations: CD14 + HLA-DR -/low cells were considered as M-MDSC-like cells ( A , B ) and CD14 + HLA-DR hi cells were identified as putative inflammatory macrophages ( C ). The lack of CD15 expression in CD14 + cells (á′′, b′′′, ć′′) corroborated the exclusive presence of M-MDSC-like cells in MS lesions. D - G : Both CD14 + cell subpopulations were stained for CD11b, confirming their myeloid cell nature. Arrowheads point to myeloid cells with M-MDSC phenotype, i.e CD14 + CD11b + HLA-DR low , while arrows indicate CD11b + inflammatory macrophages. H - O : TMEM119 + cells with microglial morphology were detected in the NAWM ( H - K ). The lack of TMEM119 immunoreactivity observed in amoeboid CD14 + HLA-DR -/low M-MDSC-like cells (pointed by arrows on L-O) confirmed the peripheral origin of these cells. Yellow and white arrowheads in L-O point to inflammatory monocyte-derived macrophages (CD14 + HLA-DR hi ) and microglial-derived macrophages (CD14 - HLA-DR hi ), respectively. P : Quantification of the density of putative M-MDSC in all MS lesion types from SPMS and PPMS patients. The abundance of CD14 + CD15 - HLA-DR -/low cells was significantly higher in the AL and in the rAIL (n=46 AL, 27 AIL and 24 IL from 33 MS patients). Q: AL and the rAIL from SPMS and PPMS patients showing a greater presence of M-MDSC-like cells than in cAIL and IL (n = 24 AL, 14 AIL and 14 IL from 20 SPMS patients; 22AL, 12 AIL and 10 IL from 13 PPMS patients). Scale bar: A-C = 5 µm; insets in A-C = 10 µm; D-G = 40 µm; H-K = 20 µm; L-O = 37 µm. Comparative analyses were carried out with ANOVA and the mean + SEM data are shown: *, # p < 0.05. In Q, * represents the difference with respect to AL and # with respect to the rAIL. rAIL, rim of AIL; cAIL, center of AIL; High. Inf Area, high inflammatory area; Weak Inf Area, weak inflammatory area.

Article Snippet: Slides were washed in TBS before incubation with mouse anti-HLA-DR antibody (1:200; Agilent, M0746, TAL.1B5 clone) and sheep anti-CD14 antibody (1:50; R&D, BAF383) in blocking solution overnight at 4°C.

Techniques: Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-L: The highest density of M-MDSC was always seen in high inflammatory areas, independent of the disease duration (long DD in A - C , short DD in G - I ). Both the cAIL and IL from SPMS with a long ( D - F ) and short disease duration ( J - L ) have a low density of M-MDSC. The arrows indicate M-MDSC (CD14 + CD15 - HLA-DR -/low ). M : Quantification of the M-MDSC distribution showing that the disease duration does not affect their density, which was always significantly higher in high inflammatory areas. N: Ratio between CD14 + HLA-DR hi cells/M-MDSCs (also referred as IIR in the main text) was significantly increased in high inflammatory areas of SPMS, independently on the disease duration. N = 11 SPMS with short DD and 9 SPMS with long DD. Scale bar: A-L = 20 µm. A comparative analysis was carried out with a Student’s t test, the means (± SEM) data are indicated: *p < 0.05, **p < 0.01 and ***p < 0.001. DD, disease duration. IIR: inflammatory-immunoregulatory ratio.

Article Snippet: Slides were washed in TBS before incubation with mouse anti-HLA-DR antibody (1:200; Agilent, M0746, TAL.1B5 clone) and sheep anti-CD14 antibody (1:50; R&D, BAF383) in blocking solution overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A: Representative flow cytometry plots for M-MDSCs in human PBMC samples. In both control and MS patients, live MNCs were gated after removing cellular aggregates and death cells based in Zombi-NIR expression. Next, immature myeloid cells were gated from monocyte subpopulation as CD33 + HLA-DR −/low populations. CD33 + HLA-DR −/low cells were assessed for CD14 and CD15 expression to identify the M-MDSC subset defined as CD14 + CD15 - (in this panel, the percentage of M-MDSCs refers to MNCs). B : The abundance of M-MDSCs measured in MS patients at an early stage of their disease course seemed to be higher than controls, although no significant differences were seen. C: The M-MDSC load in human peripheral blood was inversely correlated with the time elapsed from the first relapse to sampling. D : M-MDSCs were more abundant in MS patients at the time of their first referred relapse (MS-R, <30 days after the relapse) than in controls. E-F: In the sub-cohort of MS patients in relapse who were followed up for one year, the abundance of M-MDSCs at baseline was inversely correlated with the EDSS at baseline ( E ) and with the EDSS one year later ( F ). G : MS patients at relapse with full recovery (with an EDSS of 0 at 12 months) had a higher proportion of M-MDSCs than those with partial recovery. For correlation analysis a Spearman test was carried out (Control in B n= 26; MS in B and C n= 39; MS ≤30 days in D-G n = 23; MS >30 days in D n = 16; Total recovery n = 14 and partial recovery n = 9 MS patients in G). Comparative analysis in D was carried out with ANOVA and Student’s t test was used to compare groups of patients in B and G. The mean (± SEM) are represented, at *p < 0.05.

Article Snippet: Slides were washed in TBS before incubation with mouse anti-HLA-DR antibody (1:200; Agilent, M0746, TAL.1B5 clone) and sheep anti-CD14 antibody (1:50; R&D, BAF383) in blocking solution overnight at 4°C.

Techniques: Flow Cytometry, Expressing, Sampling

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-C: CD14 or CD15 staining was not detected in control human tissue. D-O: CD14 + cells were located both in the plaque of AL ( D , E ) and in the rim of AIL ( G, H ). By contrast, these cells were almost absent in the center or plaque of AIL ( G , H ) and IL ( J, K ). CD15 staining was not detected in any region of the MS lesions ( F , I , L ), although CD15 + granulocytes were clearly detected in the perivascular area of blood vessels ( M - O ). EC, eriochrome cyanine; AL, active lesions; AIL, mixed active/inactive lesions; IL, inactive lesions; Pl, plaque; NAWM, normal appearing white matter. Scale bar: A-C, J-N = 100 µm; D-I = 125 µm; O = 15 µm.

Article Snippet: Subsequently, immunohistochemistry (IHC) or immunofluorescence (IF) staining was performed by incubating sections overnight at 4°C with the following primary antibodies: rabbit anti-CD11b (1:100; Abcam, ab133357); rabbit anti-TMEM119 (1:50; Sigma-Aldrich, HPA0518870); biotinylated sheep anti-CD14 (1:25; R&D, BAF383), mouse anti-CD15 (1:25; Agilent, ISO62, carb-3 clone) and mouse anti-human leukocyte antigen (HLA)-DR (1:200 for IHC, 1:100 for IF; Agilent, M0746, TAL.1B5 clone).

Techniques: Staining

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-C: CD14 + cells showed different levels of HLA-DR in demyelinating lesions. Based on the intensity of HLA-DR (á′, b′′, ć′) we identified two cell subpopulations: CD14 + HLA-DR -/low cells were considered as M-MDSC-like cells ( A , B ) and CD14 + HLA-DR hi cells were identified as putative inflammatory macrophages ( C ). The lack of CD15 expression in CD14 + cells (á′′, b′′′, ć′′) corroborated the exclusive presence of M-MDSC-like cells in MS lesions. D - G : Both CD14 + cell subpopulations were stained for CD11b, confirming their myeloid cell nature. Arrowheads point to myeloid cells with M-MDSC phenotype, i.e CD14 + CD11b + HLA-DR low , while arrows indicate CD11b + inflammatory macrophages. H - O : TMEM119 + cells with microglial morphology were detected in the NAWM ( H - K ). The lack of TMEM119 immunoreactivity observed in amoeboid CD14 + HLA-DR -/low M-MDSC-like cells (pointed by arrows on L-O) confirmed the peripheral origin of these cells. Yellow and white arrowheads in L-O point to inflammatory monocyte-derived macrophages (CD14 + HLA-DR hi ) and microglial-derived macrophages (CD14 - HLA-DR hi ), respectively. P : Quantification of the density of putative M-MDSC in all MS lesion types from SPMS and PPMS patients. The abundance of CD14 + CD15 - HLA-DR -/low cells was significantly higher in the AL and in the rAIL (n=46 AL, 27 AIL and 24 IL from 33 MS patients). Q: AL and the rAIL from SPMS and PPMS patients showing a greater presence of M-MDSC-like cells than in cAIL and IL (n = 24 AL, 14 AIL and 14 IL from 20 SPMS patients; 22AL, 12 AIL and 10 IL from 13 PPMS patients). Scale bar: A-C = 5 µm; insets in A-C = 10 µm; D-G = 40 µm; H-K = 20 µm; L-O = 37 µm. Comparative analyses were carried out with ANOVA and the mean + SEM data are shown: *, # p < 0.05. In Q, * represents the difference with respect to AL and # with respect to the rAIL. rAIL, rim of AIL; cAIL, center of AIL; High. Inf Area, high inflammatory area; Weak Inf Area, weak inflammatory area.

Article Snippet: Subsequently, immunohistochemistry (IHC) or immunofluorescence (IF) staining was performed by incubating sections overnight at 4°C with the following primary antibodies: rabbit anti-CD11b (1:100; Abcam, ab133357); rabbit anti-TMEM119 (1:50; Sigma-Aldrich, HPA0518870); biotinylated sheep anti-CD14 (1:25; R&D, BAF383), mouse anti-CD15 (1:25; Agilent, ISO62, carb-3 clone) and mouse anti-human leukocyte antigen (HLA)-DR (1:200 for IHC, 1:100 for IF; Agilent, M0746, TAL.1B5 clone).

Techniques: Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A-L: The highest density of M-MDSC was always seen in high inflammatory areas, independent of the disease duration (long DD in A - C , short DD in G - I ). Both the cAIL and IL from SPMS with a long ( D - F ) and short disease duration ( J - L ) have a low density of M-MDSC. The arrows indicate M-MDSC (CD14 + CD15 - HLA-DR -/low ). M : Quantification of the M-MDSC distribution showing that the disease duration does not affect their density, which was always significantly higher in high inflammatory areas. N: Ratio between CD14 + HLA-DR hi cells/M-MDSCs (also referred as IIR in the main text) was significantly increased in high inflammatory areas of SPMS, independently on the disease duration. N = 11 SPMS with short DD and 9 SPMS with long DD. Scale bar: A-L = 20 µm. A comparative analysis was carried out with a Student’s t test, the means (± SEM) data are indicated: *p < 0.05, **p < 0.01 and ***p < 0.001. DD, disease duration. IIR: inflammatory-immunoregulatory ratio.

Article Snippet: Subsequently, immunohistochemistry (IHC) or immunofluorescence (IF) staining was performed by incubating sections overnight at 4°C with the following primary antibodies: rabbit anti-CD11b (1:100; Abcam, ab133357); rabbit anti-TMEM119 (1:50; Sigma-Aldrich, HPA0518870); biotinylated sheep anti-CD14 (1:25; R&D, BAF383), mouse anti-CD15 (1:25; Agilent, ISO62, carb-3 clone) and mouse anti-human leukocyte antigen (HLA)-DR (1:200 for IHC, 1:100 for IF; Agilent, M0746, TAL.1B5 clone).

Techniques:

Journal: bioRxiv

Article Title: Myeloid-Derived Suppressor Cells are relevant factors to predict the severity of multiple sclerosis

doi: 10.1101/2022.04.20.488896

Figure Lengend Snippet: A: Representative flow cytometry plots for M-MDSCs in human PBMC samples. In both control and MS patients, live MNCs were gated after removing cellular aggregates and death cells based in Zombi-NIR expression. Next, immature myeloid cells were gated from monocyte subpopulation as CD33 + HLA-DR −/low populations. CD33 + HLA-DR −/low cells were assessed for CD14 and CD15 expression to identify the M-MDSC subset defined as CD14 + CD15 - (in this panel, the percentage of M-MDSCs refers to MNCs). B : The abundance of M-MDSCs measured in MS patients at an early stage of their disease course seemed to be higher than controls, although no significant differences were seen. C: The M-MDSC load in human peripheral blood was inversely correlated with the time elapsed from the first relapse to sampling. D : M-MDSCs were more abundant in MS patients at the time of their first referred relapse (MS-R, <30 days after the relapse) than in controls. E-F: In the sub-cohort of MS patients in relapse who were followed up for one year, the abundance of M-MDSCs at baseline was inversely correlated with the EDSS at baseline ( E ) and with the EDSS one year later ( F ). G : MS patients at relapse with full recovery (with an EDSS of 0 at 12 months) had a higher proportion of M-MDSCs than those with partial recovery. For correlation analysis a Spearman test was carried out (Control in B n= 26; MS in B and C n= 39; MS ≤30 days in D-G n = 23; MS >30 days in D n = 16; Total recovery n = 14 and partial recovery n = 9 MS patients in G). Comparative analysis in D was carried out with ANOVA and Student’s t test was used to compare groups of patients in B and G. The mean (± SEM) are represented, at *p < 0.05.

Article Snippet: Subsequently, immunohistochemistry (IHC) or immunofluorescence (IF) staining was performed by incubating sections overnight at 4°C with the following primary antibodies: rabbit anti-CD11b (1:100; Abcam, ab133357); rabbit anti-TMEM119 (1:50; Sigma-Aldrich, HPA0518870); biotinylated sheep anti-CD14 (1:25; R&D, BAF383), mouse anti-CD15 (1:25; Agilent, ISO62, carb-3 clone) and mouse anti-human leukocyte antigen (HLA)-DR (1:200 for IHC, 1:100 for IF; Agilent, M0746, TAL.1B5 clone).

Techniques: Flow Cytometry, Expressing, Sampling