anti cd89  (Bio-Rad)

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) SDS-PAGE analysis of rLILRA5-His, used for immunisation of Balc/c mice. (B) Binding of anti-LILRA5 P4-11A mAb to magnetic beads coated with recombinant LILR or control proteins. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. Mean ± SD of n = 3 independent experiments is shown. Paired t -test between anti-LILRA5 P4-11A and isotype control, where * p < 0.05. (C) U937 cells transfected with DNA vectors expressing LILRA5 protein, or control cells, were analysed for binding of anti-LILRA5 clone P4-11A mAb. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments, one representative experiment is shown. (D) A schematic of the LILRA5CD3ζ reporter 2B4 T cell line, expressing a surface protein composed of extracellular and transmembrane LILRA5 domains fused to the cytoplasmic tail of CD3ζ. The cross-linking of CD3 or the fusion LILRA5CD3ζ protein induces phosphorylation of the ITAM (immunoreceptor tyrosine-based activation motif) domains in CD3ζ by Src kinases, and a signalling cascade that activates the NFAT (nuclear factor of activated T-cells) transcription factor. NFAT subsequently induces the expression of green fluorescent protein (GFP). (E) Cross-linking capacity of plate-bound anti-LILRA5 P4-11A mAb assessed using a LILRA5CD3ζ reporter cell line. GFP expression by LILRA5CD3ζ+ 2B4T cells or control 2B4T cells was assessed by flow cytometric analysis after incubation in wells containing plate-bound anti-LILRA5 P4-11A or isotype mAb. The % of GFP+ cells were quantified. Mean ± SD of n = 4 independent experiments are shown. One-way ANOVA, where **** p < 0.0001. (F) Development of an ELISA for detecting sLILRA5. anti-LILRA5 P4-11A mAb or isotype control were used to capture rLILRA5-His. A titration curve ( n = 3 ) of rLILRA5-His detected by anti-LILRA5 P4-11A mAb is shown.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: SDS Page, Binding Assay, Magnetic Beads, Recombinant, Control, Transfection, Expressing, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Titration

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) SDS-PAGE analysis of recombinant (r) proteins, used for assessing the specificity of anti-LILRA5 P4-11A mAb. (B) Specificity of anti-LILRA5 P4-11A antibody for LILRA5. Magnetic beads coated with rLILR or control proteins and analysed for binding of anti-LILRA5 P4-11A mAb. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments, one representative experiment is shown. (C) Concentration-dependent binding of anti-LILRA5 P4-11A mAb to rLILRA5-coated dynabeads. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: SDS Page, Recombinant, Magnetic Beads, Control, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) The normalised transcripts per million (nTPM) from the HPA dataset and the Monaco dataset are shown. nTPM values give a quantification of the transcript abundance which is comparable across samples and genes. nTPM values for classical monocytes that have been characterised to express LILRA5 is shown as a control. Mean ± SD are shown. (B) Representative example showing the gating strategy used to identify granulocyres in human whole blood using anti-CEACAM8 (CC8; clone 6/40c) and anti-LILRA5 (clone P4-11A). A first gate was set on physical parameters of SSC-A vs. FSC-A, then on SSC-A and SSC-H to eliminate doublets, then granulocytes and monocytes were gated on CD14+ and CEACAM8+, then on CEACAM8+ events to identify granulocytes. (C, D and E) Expression of LILRA5 on human monocytes and neutrophils analysed by flow cytometry analysis. C shows a representative FACS staining indicating LILRA5 expression in neutrophils and monocytes from a healthy donor. D and E show quantification of the geometric mean of monocytes ( n = 8 independent donors) and neutrophils ( n = 12 independent donors) stained with anti-LILRA5 clone P4-11A or isotype control, respectively. Paired t-test, where ** p < 0.01 and *** p < 0.001.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Control, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) Representative example showing the gating strategy used to identify monocytes in human whole blood using anti-CD14 and anti-LILRA5 (clone P4-11A). A first gate was set on physical parameters of SSC-A vs. FSC-A, then on SSC-A and SSC-H to eliminate doublets (not shown, but similarly presented in ), then monocytes and granulocytes were gated on CD14+ and CEACAM8+ (not shown), then on CD14+ events to identify monocytes. (B) Representative flow cytometry histogram showing LILRA5 expression on CD14+ monocytes. (C) Representative flow cytometry histogram showing LILRA5 expression on CEACAM8+ neutrophils.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A and B) Stimulating LILRA5 on PBMCs induces reactive oxygen species (ROS) production, as measured using Amplex Red. A representative plot is shown in A. Mean ± SD of n = 7 independent donors is shown in B. A paired-sample t -test was used to compare Area Under the Curve (AUC) after stimulating PBMCs with anti-LILRA5 P4-11A or isotype IgG1 control, where * p < 0.05. (C and D) Stimulating LILRA5 on neutrophils induces ROS production, as measured using Amplex Red. A representative plot is shown in C. Mean ± SD of n = 8 independent donors is shown in D. A paired-sample t -test was used to compare AUC after stimulating neutrophils with anti-LILRA5 P4-11A or isotype IgG1 control, where * p < 0.05.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Control

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) LILRA5 expression in whole blood from healthy donors after 4-hour ex vivo infection with E. coli ( n = 5, control n = 8) or S. aureus ( n = 5, control n = 8) (GSE65088). Mean ± SD are shown. Statistics tested by limma adjusted *** p < 0.001. (B) Expression of LILRA5 on human monocytes after ex vivo infection of whole blood by E. coli or S. aureus , analysed by flow cytometry analysis. One-way ANOVA was used to compare the mean fluorescence intensity (MFI) from anti-LILRA5 P4-11A staining (after subtracting of MFI isotype IgG1) for infection vs control, where ** p < 0.01 and * p < 0.05, is shown for n = 3 independent donors. (C) Surface LILRA5 expression on human monocytes from healthy donors ( n = 8) and sepsis patients ( n = 26), upon hospital admission, analysed by flow cytometry analysis. Each data point represents the MFI from anti-LILRA5 P4-11A stained cells after subtracting the MFI of isotype IgG1 stained cells. Mean ± SD are shown. (D) Surface LILRA5 expression on human monocytes from sepsis patients ( n = 8) at day 1, day 7 and day 14 of hospital admission, analysed by flow cytometry analysis. Each data point represents the MFI from anti-LILRA5 P4-11A stained cells after subtracting the MFI of isotype IgG1 stained cells. (E) Comparison of sLILRA5 in serum from sepsis patients ( n = 128) or healthy donors ( n = 60). Mean ± SD are shown. Statistics were tested by student t-test, where **** p < 0.0001.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Expressing, Ex Vivo, Infection, Control, Flow Cytometry, Fluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) LILRA5 expression by monocytes purified from healthy donors that were cultured ± LPS for 18 hours (GSE147310). Data from n = 5 donors are shown. Statistics tested by limma , where adjusted * p < 0.05. (B) Surface LILRA5 expression on human monocytes purified from healthy donors ( n = 3), analysed by flow cytometry analysis. Each data point represents the signal of anti-LILRA5 P4-11A relative to isotype IgG1 control. Statistics tested by paired t-test, where * p < 0.05. (C) sLILRA5 in culture supernatants from PBMCs purified from healthy donors ( n = 4), after 18 hours culture ± LPS. Statistics were tested by paired t-test, where * p < 0.05. (D) Production of reactive oxygen species (ROS) by PBMCs in response to LILRA5 ± LPS stimulation, as measured using Amplex Red. ROS production was quantified as the area under the curve (AUC). The ROS production induced by anti-LILRA5 P4-11A relative to IgG1 was calculated. Data is shown from n = 5 independent donors. Paired t -test was relative ROS production induced by anti-LILRA5 P4-11A for control vs LPS-treated cells, where ** p < 0.01.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Expressing, Purification, Cell Culture, Flow Cytometry, Control