Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: Lin- c-Kit+ progenitors in bone marrow express TLRs and their co-receptors.(A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin- c-Kit+ bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLRs and related molecules are expressed by hematopoietic stem cells and progenitor cells.(A) Lineage marker negative cells were enriched from bone marrow suspensions before staining with antibodies to hematopoietic subsets and TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the appropriate isotype matched Abs. The results shown are representative of three independent experiments. (B) Total RNA was extracted from each progenitor subset and semi-quantitative RT-PCR was conducted to detect mRNA encoding TLR4, MD-2, TLR2, and CD14. The results are shown as values normalized to peak expression for each of the transcripts and representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Marker, Staining, Quantitative RT-PCR, Expressing

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation causes a progression of Lin+ cells from LKS+ cells and soluble CD14 augments the acquisition of Mac-1 and F4/80.(A) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin+ or Lin- cells are indicated. (B) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam3CSK4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam3CSK4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures (*P<0.001). The results are representative of three independent experiments. (D) Left, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with 1 μg/ml LPS (Left, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles). After 24, 48, or 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. The graph depicts cell yields and the results are representative of two independent experiments. Right, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Right, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of Mac-1 and F4/80. The graph depicts cell yields and the data are representative of two independent experiments. (E) Sorted Flk-2- or Flk-2+ LKS+ cells (10,000 cells/well) were cultured with medium alone, LPS (10 μg/ml) or Pam3CSK (1 μg/ml). Flk-2- LKS+ cells were cultured with SCF. Flk-2 LKS+ cells were cultured with SCF and FL. After 72 h in culture, cells were analyzed by flow cytometry for expression of lineage markers (left panels). Percentages given in quadrants indicate the frequencies of Lin+ or Lin- cells and the bar graphs on the right depict cell yields. The data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments. (*P<0.03: Flk-2- LKS+, *P<0.002: Flk-2+ LKS+)

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Flow Cytometry, Expressing, Recombinant

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis.(A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88-/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam3CSK4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19+ cells or Mac-1+ cells (Right) and the data represent mean values with standard deviations from triplicate cultures (*P<0.02). The results are representative of those obtained in five independent experiments. (B) Subsets of the recovered cells described in panel (A) were sorted and used to prepare Giemsa-May-Grünwal stained slides. (C) Cultured cells from C57BL/6 mice were also analyzed by flow cytometry for expression of Gr-1 and CD11c. (D) Sorted CLPs from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with a range of concentrations of LPS or combinations of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graph depicts cell yields for CD19+ cells or Mac-1+ cells (Right). Data represent mean values with standard deviations from triplicate cultures (*P<0.003). The results are representative of three independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Staining, Cell Culture, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease.

doi: 10.4049/jimmunol.1000536

Figure Lengend Snippet: FIGURE 6. rCRT/39–272 activates murine B cells and macrophages through the CD14/TLR4 pathway. Freshly purified splenic B cells (4 3 105 cells/ well) from C3H/HeN and C3H/HeJ mice were stimulated, in triplicates, with rCRT/39–272 at various concentrations or with LPS (3 mg/ml) in 96-well plates for 72 h. During the last 8 h of culture, the cells were pulsed with [3H]TdR, and the results are expressed as [3H]TdR incorporation (cpm) 6 SD (A). Freshly prepared peritoneal macrophages (1 3 105 cells/well) from C3H/HeN and C3H/HeJ mice were also stimulated with rCRT/39–272 (0.3–10 mg/ml), LPS (3 mg/ml), or peptidoglycan (20 mg/ml) in 96-well plates for 48 h. Concentration of TNF-a in the culture supernatant was determined using an ELISA kit, and the results are expressed as mean 6 SD (B). Modified CRT-based ELISA was performed for identification of CRT-specific ligands (C), in which plate-bound rCRT/39–272 was allowed to react with recombinant soluble fusion protein mCD14-hIgG1-Fc (rmCD14), mCD40-hIgG1-Fc (rmCD40), or mBAFFR-hIgG1-Fc (rmBAFFR) in triplicate wells for 1 h, followed by HRP-conjugated goat anti-human IgG with OPD as substrate. The results are expressed as mean OD492 6 SD. pp , 0.05; ppp , 0.01. As additional specificity controls, LPS-based (D) and CD40L-based (E) ELISAs were also carried out in similar fashion. Results are representatives of three independent experiments. PGN, peptidoglycan.

Article Snippet: Recombinant mouse CD40L and recombinant fusion proteins between mouse CD14 and human IgG1 Fc fragment (rmCD14-Fc), mouse CD40 and human IgG1 Fc (rmCD40-Fc), mouse BAFFR and human IgG1 Fc (rmBAFFR-Fc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: BMC Immunology

Article Title: LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-Ligand

doi: 10.1186/s12865-015-0067-2

Figure Lengend Snippet: Flow cytometry analysis of CD3, CD14, CD15, CD19, and CD41 expression. Platelet-rich plasma (PRP) was prepared by centrifugating peripheral blood from healthy donors and collecting in the upper phase. Leukocytes were also recovered at the interphase between the PRP and the red cells. The contaminating red cells in the leukocyte suspension were removed by lysis. Platelets (A,D) and leukocytes (C,D) were stained for lineage markers (CD3 + T cells, CD14 + monocytes, CD15 + neutrophils, CD19 + B cells and CD41+ platelets). Fluorescence analysis was gated on the R5 region for PRP (A,B) and on R6 region of the cytogram for leukocytes (C,D) . The presence of mononuclear cells in PRP preparation, although significant, was clearly lower than in leukocyte preparation. The expression of CD14 on platelets from PRP was compared to that of washed platelets (E) after gating on CD41-positive cells in the R5 region of the cytogram (data are expressed as percentage of CD expression ± SD; n = 10 experiments). Cytograms shown (A,C) are representative of 10 independent experiments. *P < 0.05 (Wilcoxon paired test; platelets PRP vs. washed platelet suspension).

Article Snippet: Platelets (3 × 10 8 ) were stimulated for 30 min at room temperature (RT) with ultrapure S-LPS from E. coli 0111: B4 (1–10 μg/ml; Cayla-Invivogen, Toulouse, France) and recombinant CD14 (0.25 μg/ml; R&D Systems Europe Ltd, Lille, France).

Techniques: Flow Cytometry, Expressing, Clinical Proteomics, Suspension, Lysis, Staining, Fluorescence

Journal: BMC Immunology

Article Title: LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-Ligand

doi: 10.1186/s12865-015-0067-2

Figure Lengend Snippet: Flow cytometry analysis of CD63 expression on CD41 + platelets. Platelets were or stimulated or not with TRAP (50 μg/ml, 30 min at RT). Platelets from platelet-rich plasma (PRP; black bars), washed (PBS) platelet suspensions (khaki bars) or washed (PBS) platelet suspensions with added recombinant soluble CD14 (white bars) were tested. Platelets were then stimulated, or not, with E. coli LPS (1–10 μg/ml; 30 min at RT), stained with allophycocyanin-conjugated anti-CD41 and phycoerythrin-conjugated anti-CD63 mAbs. Gating was made on the platelet population, (R5 region according to FSC/SSC comparable to the Figure cytogram). Double positive cells (CD41 + CD63+) were then measured. Data are expressed as fold increase of controls (± SD; n = 10 independent experiments). * P < 0.05 (Wilcoxon paired test; stimulated vs. non-stimulated).

Article Snippet: Platelets (3 × 10 8 ) were stimulated for 30 min at room temperature (RT) with ultrapure S-LPS from E. coli 0111: B4 (1–10 μg/ml; Cayla-Invivogen, Toulouse, France) and recombinant CD14 (0.25 μg/ml; R&D Systems Europe Ltd, Lille, France).

Techniques: Flow Cytometry, Expressing, Clinical Proteomics, Recombinant, Staining

Journal: BMC Immunology

Article Title: LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product, soluble-CD40-Ligand

doi: 10.1186/s12865-015-0067-2

Figure Lengend Snippet: Platelet sCD40L production and secretion. Platelets were unstimulated or stimulated with TRAP (50 μg/ml, 30 min at RT). Platelets from platelet-rich plasma (PRP; black bars), or from platelet suspensions with added anti-human CD14 (clone 18D11 20 μg/ml; grey bars) or isotype control (clone 15H6; white bars) were tested. Platelets were unstimulated or stimulated with E. coli LPS (1–10 μg/ml; 30 min at RT). Mean sCD40L release was 1.2 ng/ml for unstimulated PRP and 1.5, 1.9, 2.2 and 3.1 ng/ml for LPS stimulation at concentrations of 1, 2, 3 and 10 μg/ml, respectively. Data are expressed as fold increase of controls (± SD; n = 10 independent experiments). * P < 0.05 (Wilcoxon paired test; stimulated vs. non-stimulated).

Article Snippet: Platelets (3 × 10 8 ) were stimulated for 30 min at room temperature (RT) with ultrapure S-LPS from E. coli 0111: B4 (1–10 μg/ml; Cayla-Invivogen, Toulouse, France) and recombinant CD14 (0.25 μg/ml; R&D Systems Europe Ltd, Lille, France).

Techniques: Clinical Proteomics, Control

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A, B Stimulated or unstimulated MDM were stained for classical macrophage markers (CD68, CD14) and M1 and M2 macrophage markers (CD163, CD80, CD86 and CD40) and analysed by FACS.

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: A schematic representation of our experimental approach showing rTLR4 conjugation to magnetic nanoparticles and its in vitro complexation with LPS in the presence of free forms of MD2 and CD14 co-receptors. The bottom left panel shows the actual optical micrographs of MP-rTLR4-MD2-LPS (test) and MP-rTLR4 (control) complexes recorded in brightfield and complementary fluorescence modes at two different emission wavelengths: 525 nm (green) for alexafluor-tagged LPS and 647 nm (red) for alexafluor-tagged MD2.

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Conjugation Assay, In Vitro, Fluorescence

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: (A) LPS monomerization aided by surfactant Tween 20 addition. (B) The importance of CD14 and MD2 on the extent of complexation, and (C) the molar amounts of molecules used in these reactions. (D) Cross-validation of LPS capture using the LAL assay. (E–H) Sequential scouting experiments: Bulk concentrations of LPS (E) , rTLR4 (F) , MD2 (G) , and CD14 (H) were optimized one-by-one and the optimized values were fixed in all the subsequent experiments to get maximum LPS capture efficiency at each step. The fixed concentration values of the remaining reagents (all in nM) are listed on top of each graph. All experiments were performed in 0.5% PBST, pH 7.4 ( n = 3) (SN = supernatant, ns, p >0.05, * p = 0.01–0.05, ** p = 0.001–0.01, *** p < 0.001).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: LAL Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: (A) Time-lapsed measurements of LPS, MD2, and CD14 concentrations showing molecular binding kinetics and the role of CD14 as a catalyst. While the LPS and MD2 molecules were measured using their fluorescence intensity in the SN, CD14 was quantified via silver staining. (B) CD14 visualized after alexafluor tagging (λ em = 647 nm). The complementary brightfield and fluorescence optical images showed CD14 in the bound state only in the middle of the reaction cycle. (C) Varying the sequence of molecular addition had little impact on the extent of LPS capture. Here, sequential addition implies prior incubation of CD14 with LPS and MP-rTLR4 with MD2 followed by their rapid mixing as opposed to co-incubation of all the reagents from the beginning ( n = 3) (SN = supernatant).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Binding Assay, Fluorescence, Silver Staining, Sequencing, Incubation

Journal: Frontiers in Immunology

Article Title: A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

doi: 10.3389/fimmu.2020.00005

Figure Lengend Snippet: Inhibitory ligand screening. Results shown for LPS-interacting alexidine and sushi molecules used as test, and LPS-non-interacting bacitracin and vancomycin molecules used as control ( n = 3). While the LPS fluorescence intensity in the SN remained constant upon addition of MP-rTLR4-MD2-CD14 assay mix (marked here as “A”) in the presence of alexdine and sushi, it reduced significantly with bacitracin and vancomycin suggesting the inhibitory potential of the first two molecules for obstructing rTLR4 pathway. All the data have been normalized with respect to the fluorescence intensity before “A” addition. The concentrations of each ligand used in reaction was 2.4 μM in their respective reactions (SN = supernatant, ** p = 0.001–0.01, *** p < 0.001).

Article Snippet: Human recombinant toll like receptor 4 (rTLR4) (R & D Systems, USA); Recombinant human myeloid differentiation-2 (MD2) protein (Abcam, USA); His-tagged human cluster differentiation-14 (CD14) protein (Sino Biologicals, USA); Sushi peptide of purity > 95% [HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC (M.W.

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade

doi: 10.1038/ncomms14979

Figure Lengend Snippet: ( a ) Bright field (BF) images of co-cultures of EMT6 tumour cells (TC) (black arrow) with mMDSC (white arrows) or gMDSCs (red arrows) derived from 4T1 tumour-bearing mice. Morphological characteristic of EMT phenotype is visibly induced by the mMDSCs (top panel) which also show a strong affinity towards tumour cells. ( b – e ) mMDSC-induced EMT phenotype is assessed by enhanced expression of Vimentin and CK14. ( f ) Enhanced expressions of Vimentin and Twist induced by the mMDSCs from 4T1 tumour-bearing mice are verified by qPCR analyses. ( g – j ) mMDSCs from 4T1 tumour-bearing mice induce tumour cell invasion and CSC phenotype as shown by CD24-CD29 phenotype and tumour sphere forming assay. ( k , l ) In situ analyses of primary tumour and metastatic lesions by immunofluorescence staining reveal mMDSCs at the invasive edge of the primary tumour (TC, tumour center; TE, tumour edge) and gMDSCs around the pulmonary metastatic lesions in 4T1 tumour-bearing BALB/c mice (Met, metastasis; Inft, infiltrates). ( m ) Substantially higher number CD14-positive human mMDSCs were detected in metastatic human breast cancers compared to the indolent tumours. Results are presented as mean±s.d. ( n =3). Scale bar, 50 μm; * P <0.05, ** P <0.005; unpaired t -test.

Article Snippet: The following primary antibodies were used: Ly6C (#ab15627-Dilution 1/100, Abcam), Ly6G (#MAB1037-Dilution 1/100, R&D biosystem), Ki67 (#12202-Dilution 1/100, Cell Signaling), Vimentin (#550513 Dilution 1/100, BD Biosciences), CD14 (#10073-H08H Dilution 1/100, Sino biological).

Techniques: Derivative Assay, Expressing, In Situ, Immunofluorescence, Staining