anti-cd33m (Becton Dickinson)

Becton Dickinson anti-cd33m

Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to <t>biotin-CD33M</t> (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Anti Cd33m, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-cd33m - by Bioz Stars, 2026-10

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cd33 (m)-pr (Santa Cruz Biotechnology)

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Gene Silencers generally consist of pools of three to five target-specific 19-25 nucleotide sequences in length. For independent verification of CD33 gene silencing results, individual duplex components or plasmids are also available upon request. Suitable

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Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing

C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Techniques: Binding Assay, Staining, Flow Cytometry, Isolation, Two Tailed Test

Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Techniques: Expressing, Flow Cytometry, Isolation, Two Tailed Test

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