Journal: Oncology Reports

Article Title: Kevetrin induces apoptosis in TP53 wild-type and mutant acute myeloid leukemia cells

doi: 10.3892/or.2020.7730

Figure Lengend Snippet: Effect of continuous kevetrin treatment on viability and apoptosis induction in AML cell lines and primary samples. (A and B) Viability of TP53 -wt OCI-AML3 and MOLM-13 and TP53 -mut KASUMI-1 and NOMO-1 cell lines treated with different concentrations of kevetrin (85, 170 and 340 µM) for 24 and 48 h. (C and D) Quantification of Annexin V + cells in AML cell lines treated with kevetrin at different concentrations (85, 170 and 340 µM) for 24 and 48 h. (E and F) Cell cycle analysis of cell lines treated with 340 µM kevetrin for 24 and 48 h. Values represent the mean ± standard deviation of three biological replicates. (G) Cell viability and (H) percentage of Annexin V + cells in bone marrow mononuclear cells from AML patients exposed to increasing concentrations of kevetrin. (I) Percentage of AML blasts relative to control. Values represent the mean ± standard deviation. Symbols indicate samples from AML patients (no. 01: TP53 -mut AML; nos. 02, 03 and 04: TP53 -wt AML). (J) Percentage of Annexin V − cells in AML blasts, monocytes and lymphocytes. Blasts were defined as CD33 + CD14 − cells in the CD45/side scatter ‘blast gate’, monocytes as CD33 + CD14 + , B lymphocytes as CD19 + and T lymphocytes as CD3 + . *P<0.05, **P<0.01, ***P<0.001. AML, acute myeloid leukemia; KEV, kevetrin; CTRL, control; wt, wild-type.

Article Snippet: In primary samples, Annexin V staining was combined with surface markers using the following antibodies: CD45-APC Vio770 (cat. no. 130-110-635), CD33-APC (cat. no. 130-111-020), CD14-PerCP Vio 700 (cat. no. 130-110-523), CD3-PE (cat. no. 130-113-139) (all from Miltenyi Biotec GmbH, dilution 1:50) and CD19-PE Cy7 (cat. no. 302216, 1:10, BioLegend, Inc.).

Techniques: Cell Cycle Assay, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33 reporter cell line constructs. ( a ) Schematic drawing of the two CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ΔE2 ) were fused to TYROBP/DAP12. ( b ) Schematic drawing of the readouts for the CD33 reporter cell line. CD33 can be activated by ligands or antibodies, which results in increased SYK phosphorylation and consequently increased intracellular calcium levels. ( c ) Gel electrophoresis image of CD33-DAP12 constructs cloned into pcDNA5/FRT after digestion by EcoRI. ( d ) Successful exchange of the viral CMV promoter with the human EEF1A1 promoter was indicated by a second band after digestion with XhoI at 1,339 bp (CD33M) or 958 bp (CD33 ΔE2 ). ( e ) Gel electrophoresis image of pcDNA5/FRT-CD33-DAP12-GCaMP6m plasmids after digestion with XhoI. GCaMP6m positive clones exhibited three bands compared to the control (Ctrl) with only two bands. Gel images were cropped for better visualization. Supplementary Fig. shows the uncropped full-length gel.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Construct, Binding Assay, Phospho-proteomics, Nucleic Acid Electrophoresis, Clone Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33-specific and control antibodies.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Imaging

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Flow cytometric analysis of CD33 surface expression. ( a ) Schematic drawing of the CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ∆E2 ) were fused to TYROBP/DAP12. CD33 ∆E2 can be identified by binding of the antibody clone 1c7/1 (blue) but not WM53 or P67.6 (red), whereas all three antibody clones can bind CD33M. ( b ) The CD33-DAP12 and CD33-DAP12-GCaMP6m cells were stained for CD33 surface expression with the antibody clones 1c7/1, WM53 and P67.6. A representative flow cytometry histogram plot for the CD33M-DAP12-GCaMP6m cells is shown (left side). All three tested antibodies were able to stain full-length CD33 on the cell surface. Expression of variant 2 CD33 from CD33 ∆E2 -DAP12 and CD33 ∆E2 -DAP12-GCaMP6m cells was only detected by antibody clone 1c7/1. A representative flow cytometry histogram plot for the CD33 ∆E2 -DAP12-GCaMP6m cells is shown (right side). ( c ) Quantification of CD33 staining showed a high percentage of CD33 expressing cells in the CD33M-DAP12-GCaMP6m line for all three tested antibody clones but only the CD33 antibody clone 1c7/1 was able to detect CD33 in CD33 ∆E2 -DAP12-GCaMP6m expressing cells. The antibody clones WM53 and P67.6 did not show any staining of CD33 ∆E2 -DAP12 expressing cells. ( d ) Quantification of CD33 staining revealed a high percentage of cells in the CD33M-DAP12 line expressed CD33, and was detected by all three antibody clones. CD33 in CD33 ΔE2 -DAP12 expressing cells was only detected by antibody clone 1c7/1. Data are shown as mean + SEM of three to five independent experiments; *** p ≤ 0.001 compared to Secondary Control determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Expressing, Construct, Binding Assay, Clone Assay, Staining, Flow Cytometry, Variant Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: pSYK detection in CD33M-DAP12 cell lines. ( a ) pSYK detection in CD33M-DAP12 reporter cells treated with CD33-specific antibodies. Addition of CD33 antibodies P67.6 and 1c7/1 resulted in increased pSYK levels, whilst P67.6 F(ab), WM53 as well as the different isotype IgG1 control antibodies did not show any change in endogenous pSYK levels. ( b ) 1c7/1 and P67.6 dose–response curve in CD33M-DAP12 reporter cells. Addition of CD33 antibody clones 1c7/1 and P67.6 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment. Data are presented as mean ± or + SD; ** p ≤ 0.01 compared to untreated determined by one-way ANOVA analysis followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Clone Assay

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Calcium imaging in CD33-DAP12-GCaMP6m reporter cell lines. ( a ) Schematic time line of image acquisition and compound handling. ( b , c ) Calcium imaging analyzed as ΔF/F(t) in CD33-DAP12-GCaMP6m lines. Addition 100 µM dATP led to a strong increase in intracellular calcium levels in both cell lines, CD33M- and CD33 ∆E2 -DAP12-GCaMP6m, with a peak at around 20–25 s. The CD33 antibody clones 1c7/1 and P67.6 evoked a selective intracellular calcium response only in CD33M-DAP12-GCaMP6m cells. The CD33 antibody clones WM53 and P67.6 F(ab) as well as the isotype IgG1/F(ab’)2 antibodies did not show a change in intracellular calcium levels. ( d , e ) The area under the curve as well as the maximum ΔF/F(t) signal calculated from independent experiments showed a significant increase in dATP treated samples in both CD33-DAP12-GCaMP6m lines and a selective increase in CD33M-DAP12-GCaMP6m expressing cells if treated with the CD33 antibody clone P67.6 or 1c7/1. Data are presented mean + SEM; n = 3–6; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 compared to 10 µg/ml IgG1 determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Imaging, Clone Assay, Expressing

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Activation of endogenous CD33 in iPSdMiG by CD33 agonistic antibodies. ( a ) pSYK analysis in TREM2 + DAP12 reporter cells. Addition of anti-TREM2 antibody AF1828 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment only in TREM2 + DAP12 but not DAP12 expressing control reporter cells. Data are presented mean ± SD. ( b ) CD33 antibodies P67.6 and 1c7/1 were able to decrease the increased pSYK/tSYK levels triggered by TREM2 activation in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pSYK/tSYK levels after TREM2 activation. Data are presented mean + SEM; n = 3–6; ** p ≤ 0.01 compared to IgG1 (anti-CD33 Ctrl) determined by Welch ANOVA followed by Games-Howell post hoc test. ( c ) CD33 antibodies P67.6 and 1c7/1 decreased the phagocytic uptake of pHrodo S. aureus BioParticles in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pHrodo S. aureus BioParticle phagocytosis. Data are presented mean + SEM; n = 3; ** p ≤ 0.01 and * p ≤ 0.05 compared to IgG1 determined by ANOVA followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Activation Assay, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). (B) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. (C) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). (D) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. (E) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release (n = 3 per condition; values are plotted as the mean ± SD). (F) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). (G) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. (H) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). (I) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). (J) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E–J are representative of 3 independent experiments. ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (D and E) and 1-way ANOVA followed by Tukey’s test (F–J). α, anti; Max, maximum.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Staining, Flow Cytometry, Binding Assay, Liposomes, Control, Clinical Proteomics

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of GFP expression on representative peritoneal mast cells harvested from C57BL/6J, control-Tg (Mcpt5-Cre–Rosa26-Stopfl/fl-CD33+), and CD33-Tg (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+) mice. Mast cells were defined as PI–CD45+c-Kit+. Baseline GFP signal was determined by mast cells from C57BL/6J mice. (B) Quantification of the percentage of GFP+ peritoneal mast cells from mice of the 3 genotypes. Tg mice bearing 1 or 2 copies of CD33 were used. Both male and female mice 8 weeks or older were analyzed, with no difference observed. (C) Numbers of peritoneal mast cells from the same mice of the 3 genotypes as in B. (A–C) Results were compiled from 6 experiments. (D) Staining of peritoneal cells harvested from control-Tg or CD33-Tg mice with anti-CD33 (clone WM53) or isotype control, as analyzed by flow cytometry. (E) Binding of fluorescent liposome, with or without CD33L (20 μM), to peritoneal mast cells (c-Kit+FcεRI+CD45+). (F) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L. (G) Cytokine induction of CD33+ BMMCs following treatment with TNP-LP (40 μM), TNP-LP-CD33L (40 μM), LP-CD33L (40 μM), or a mixture of TNP-LP and LP-CD33L (40 μM each). Supernatant from the unstimulated cells was subtracted as a background. (H) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of anti-CD33 (2 μg/ml). (I and J) Cytokine production of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (40 μM) in the presence of anti-CD33 (10 μg/ml). Supernatant from untreated cells was subtracted as a background. Results shown are representative of 3 (D–G) or 2 (H–J) independent experiments. (F–J) Values are plotted as the mean ± SD (n = 3 per condition). ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (F) and 1-way ANOVA followed by Tukey’s test (C and G–J).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Expressing, Control, Staining, Flow Cytometry, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Phosphorylation of Syk, PLCγ1, MEK, and ERK in LAD2 cells after a 3-, 10-, or 30-minute stimulation using TNP-LP or TNP-LP-CD33L (2 μM), as evaluated by Western blotting. (B) Phosphorylation of Syk and ERK in LAD2 cells after a 10-minute stimulation using TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (C) Phosphorylation of Syk, PLCγ1, PLCγ2, JNK, AKT, and ERK in CD33+ BMMCs cells after a 10-minute stimulation with TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (A–C) Total Syk and ERK were used as loading controls. (D–F) Proposed mechanisms of IgE/FcεRI signaling induced by antigenic liposomes and recruitment of CD33 by CD33L. (D) TNP-LP stabilizes the anti–TNP-IgE–FcεRI complex in lipid rafts with Src kinases that initiate the FcεRI signaling cascade. We propose that CD33 has no basal impact on signaling, because it is not constitutively localized in the same microdomain with FcεRI. (E) TNP-LP-CD33L recruits CD33 to the anti–TNP-IgE–FcεRI immunological synapse. Our results suggest that the cytoplasmic ITIMs of CD33 were phosphorylated by Src kinases and then recruited tyrosine phosphatases such as Shp-1, which dephosphorylated Syk, and other kinases. (F) Proposed model showing that monoclonal anti-CD33 antibodies (or LP-CD33L) block recruitment of CD33 to the IgE-FcεRI complex and enable mast cell degranulation induced by TNP-LP-CD33L.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Phospho-proteomics, Western Blot, Liposomes, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: Display of CD33L on antigenic liposomes suppresses PCA and PSA in CD33-Tg mice (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+), but not in control-Tg mice (Mcpt5-Cre– Rosa26-Stopfl/fl-CD33+). Mice bearing 1 or 2 copies of the CD33 transgene were used. In I, Mcpt5-Cre+/– mice expressing human CD33 (CD33-Tg) were crossed with Ptpn6fl/fl mice to yield mice with mast cells expressing CD33 and no Shp-1 (CD33-Tg/Shp-1–KO). (A) Injection scheme for the PCA model. The genotypes of the mice were determined by PCR after the experiments. (B) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in control-Tg mice. (C) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 14) or TNP-LP-CD33L (50 μg, n = 28) in control-Tg mice. (D) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in CD33-Tg mice. (E) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 21) or TNP-LP-CD33L (50 μg, n =27) in CD33-Tg mice. (F) Injection scheme for the PSA model. (G–I) Decrease in rectal temperature induced by TNP-LP or TNP-LP-CD33L (150 μg) in control-Tg mice (G), CD33-Tg mice (H), and CD33-Tg mice lacking Shp-1 (I) that were sensitized with 10 μg anti–TNP-IgE. (G–I) Values are plotted as the mean ± SEM at the indicated time points. Data are from 1 experiment (G and I) or were compiled from 3 (H) or 9 sets of experiments (C and E). ***P < 0.001 and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s test (C and E), repeated-measures (RM) 2-way ANOVA (G and H), and RM 2-way ANOVA followed by Tukey’s test (I).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Liposomes, Control, Expressing, Injection, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). (B) Changes in rectal temperature induced by treatment or the challenges indicated in A. (C) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). (D) Rectal temperature induced by the treatment or challenge illustrated in C. (B and D) Values are plotted as the mean ± SEM. (E) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. (F) Frequencies of mast cells from peritoneal fluid from mice treated in E. Mast cell frequencies were determined by c-Kit+CD45+PI– cells. (G) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C. (H) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G. The background was determined using untreated cells from a naive mouse. (I) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 (F–H) or 3 (I) independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by RM 2-way ANOVA (B), RM 2-way ANOVA followed by Tukey’s test (D), and unpaired, 2-tailed Student’s t test (F–I).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Injection, Control, In Vitro, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of mast cells isolated from discarded human skin (c-KithiFcεRI+ gated on PI–CD45+CD3–CD19–CD56– cells) (left) and overlay of isotype control and anti-CD33 staining of gated mast cells (right). (B) MFI of antibody staining of Siglecs on mast cells isolated from skin that was discarded following surgical procedures (n = 1 to 10 donors). (C) Time course of the percentage of bronchoconstriction of hPCLSs. Lung slices were sensitized with human IgE (4 mg/ml, gray) or anti–TNP-IgE (10 μg/ml, black and red) with recombinant human SCF (200 ng/ml, R&D Systems) overnight. Slices were challenged with anti–human IgE (20 μg/ml, gray), TNP-LP (50 μM, black), or TNP-LP-CD33L (50 μM, red) over a 10-minute period. The airway luminal area over time was compared with the baseline luminal area and expressed as the percentage of bronchoconstriction. Values represent the mean ± SEM. (D) AUC induced by the indicated treatments. AUC values below 0 are plotted as 0. (E) Percentage of bronchoconstriction induced by the indicated agents at 10 minutes. (F) Following stimulation with the indicated reagents, the percentage of bronchoconstriction induced by CCh (0.1 mM) was measured. *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Tukey’s test (D–F). Gating of skin mast cells in A is representative of the 10 donors. Data in C–F were compiled from 5 or 9 lung slices from 2 donors.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Isolation, Control, Staining, Recombinant

Journal: NPJ Precision Oncology

Article Title: DDX41 facilitates PD-L1-mediated immune escape in OSCC via the phase separation and activation STING pathway

doi: 10.1038/s41698-026-01308-1

Figure Lengend Snippet: A Representative immunohistochemical staining of DDX41 in OSCC compared to adjacent normal tissues (Scale bar, 50 μm). B Quantitative tissue scoring of DDX41 protein expression in control tissues ( n = 10) and OSCC tissues ( n = 54). C Kaplan–Meier analysis shows that DDX41 expression is significantly associated with overall survival in OSCC patients (DDX41 low, n = 34; DDX41 high, n = 20; p = 0.0347) . D – G Representative immunohistochemical staining of DDX41 and p-p65/PD-L1/CD33/CD8 in OSCC (Scale bar, 50 μm). H , I Correlation of DDX41 and p-p65/PD-L1 in OSCC tissue microarrays (p-p65 p < 0.0001, r = 0.5969)/ (PD-L1 p < 0.001, r = 0.4577). J , K , N , O Quantitative tissue scoring of p-p65/PD-L1/CD33/CD8 protein expression in control tissues ( n = 10) and OSCC tissues ( n = 54). L, M Correlation of DDX41 and CD33/CD8 in OSCC tissue microarrays (CD33 p < 0.0001, r = 0.6567) / (CD8 p < 0.0001, r = 0.6099). P Heat map of the correlation of protein expression among DDX41, p-p65, CD33, PD-L1 and CD8. The quantitative data above are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The immuno-histochemical procedure involved the use of primary antibodies against DDX41 (1:200, Proteintech, 27500-1-AP), p-STING (1:200, Affinity, AF7416), p-TBK1 (1:200, Bioss, BC07208022), p-p65 (1:200, Affinity, AF2006), PD-L1 (1:200, CST, 13684 T), IFN-γ (1:200, Proteintech, 80979-1-RR), TNF-α (1:200, Proteintech, 19272-1-AP), anti-CD8a (1:150, CST, 98941), CD33 (Affinity, DF6789) and PCNA (1:200, CST, 13110 T).

Techniques: Immunohistochemical staining, Staining, Expressing, Control

Journal: Cell

Article Title: Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19

doi: 10.1016/j.cell.2020.08.002

Figure Lengend Snippet:

Article Snippet: CD33 , Fluidigm , Pro# 3163023B.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Staining, Functional Assay, Software