11-0168-41

antibody from Invitrogen Antibodies

Targeting: FCGR3B CD16, CD16b, FCG3, FCGR3

Provider product page for 11-0168-41

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Antibody Data
Antigen structure
References [38]
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Validations
Flow cytometry [1]
Other assay [25]

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Product number: 11-0168-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD16 Monoclonal Antibody (eBioCB16 (CB16)), FITC, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The eBioCB16 monoclonal antibody recognizes CD16 (Fc gammaRIII), the low-affinity receptor for IgG with an apparent molecular weight of 50-80 kDa. CD16 is represented by two similar genes, CD16A (Fc gammaRIIIA), which exists as a hetero-oligomeric polypeptide-anchored form in macrophages and NK cells and CD16B (Fc gammaRIIIB), which exist as a monomeric GPI-anchored form in neutrophils. Furthermore, there are two known polymorphisms of CD16B, NA-1 and NA-2. Individuals homozygous for NA-2 show a lower phagocytic capacity compared with NA-1. CD16 binds IgG in the form of immune complexes and shows preferential binding of IgG1 and IgG3 isotypes and minimal binding of IgG2 and IgG4. Upon IgG binding, both CD16 isoforms initiate signal transduction cascades that lead to a variety of responses including antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, degranulation and proliferation.
Conjugate: Green dye
Antibody clone number: eBioCB16 (CB16)
Concentration: 5 µL/Test

FCGR3B protein structure - 11-0168-41 shown in red.

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Supportive validation

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Experimental details: Figure 4 The IL-36 receptor is preferentially expressed by plasmacytoid dendritic cell s. ( a-e ) Representative flow cytometry plots showing IL36R surface expression compared with fluorescence minus one control. ( a ) neutrophils (gated as CD14 + , CD15 + , CD16 + cells); ( b ) innate lymphoid cells (lineage - [CD3 - , CD4 - , CD19 - , CD20 - , CD56 - ], CD127 + ); ( c ) monocytes (CD3 - , CD20 - , CD19 - , CD56 - ) separated into classical (CD16 - , CD14 high ), intermediate (CD16 + , CD14 + ), and pro-inflammatory (CD16 high , CD14 - ) populations; ( d ) pDCs (lineage - , HLA-DR + , CD123 + , CD11c - ) and mDCs (lineage - , HLA-DR + , CD123 - , CD11c + ). ( e ) Histogram showing the percentage IL36R + cells in each leukocyte population. Data were obtained in at least three GPP cases and three sex-matched controls. Results are presented as mean +- SEM. No significant differences were observed between GPP cases and healthy donors. FMO, fluorescence minus 1; IL36R, IL-36 receptor; GPP, generalized pustular psoriasis; mDC, myeloid dendritic cell; Mo, monocytes; pDC, plasmacytoid dendritic cell; SEM, standard error of the mean.
Conjugate: Green dye

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Experimental details: Figure S2. Sorting strategy and antibody sequence analysis. (A) Sorting strategy. Forward and side scatter (FSC and SSC, respectively) were used to gate on single lymphocytes. Dump channel included CD3, CD8, CD14, CD16, and a viability dye. CD20 + B cells that failed to bind OVA (OVA - ) but did bind to the TBEV EDIII bait coupled with both PE and AF647 fluorophores were purified. (B) For each donor, the number of V gene somatic nucleotide mutations is shown on the left and the amino acid length of the CDR3 is shown on the right. (C) As in B, but for all donors combined. For B and C, horizontal red lines indicate the mean. (D) Distribution of hydrophobicity GRAVY scores at the IGH CDR3 of antibodies from all donors combined and compared with human repertoire (). P < 0.0001 was determined using the Wilcoxon nonparametric test. Related to Fig. 2 , Table S2 , and Table S3 .
Conjugate: Green dye

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Experimental details: Figure 1 Metformin treated cancer cells polarized macrophage toward M1 phenotype THP-1 cells were stimulated with PMA (200 nM) for 24 h, then incubated with breast cancer (MDA-MB231/MDA-MB453) conditioned medium (CM) with or without metformin (60 muM) for 6 h, followed by flow cytometry analysis to quantify the amount of CD206, an M2 macrophage marker, and CD16, an M1 marker ( A, B ). Data are expressed as mean +- SD, * p < 0.05. DMSO: control; Met: metformin. Representative flow data shown are from experiments independently performed at least three times.
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Supportive validation

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Experimental details: Figure S1. Immunopathological characteristics of COVID-19 patients. (A) Doublet cells were excluded by forward scatter height (FSC-H) and forward scatter area (FSC-A) gating for all flow cytometry analysis. Viable cells were identified using fixable viability stain and side scatter area (SSC-A) gating. Neutrophils were identified as cells stained for CD15 + CD16 + among CD14 - CD19 - cells. (B) Flow cytometry analyses of living cells from the whole blood of healthy controls (H.Control) or COVID-19 patients. (C) Frequency and absolute numbers of CD15 + CD16 + neutrophils gated on CD14 - CD19 - live cells from whole blood from healthy controls ( n = 7) or COVID-19 patients ( n = 7). (D) CT of the chest of one patient who died from COVID-19. Images from apical to basal segments (I to IV) show multiple consolidations with air bronchograms in a peripheral and peribronchovascular distribution, more evident in the lower lobes, associated with ground-glass opacities. (E) Representative pulmonary histological findings in 10 cases, autopsied by ultrasound-guided, minimally invasive autopsy. I: The area with interstitial and alveolar neutrophilic pneumonia with diffuse alveolar damage and hyaline membranes in the alveolar space (black arrows). Septal vessel with margination of leukocytes and an intraluminal early fibrin thrombus (green star). II: Area with neutrophilic pneumonia (red arrow), septal thickening, epithelial desquamation, and squamous metaplasia (black arrows). I and II: H&
Conjugate: Green dye

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Experimental details: Figure 2. Circulating neutrophil populations are expanded in COVID-19 and non-COVID-19 ARDS. ( A ) Patient classification (healthy control, HC, non-COVID-19 ARDS, NA, moderate COVID-19, MC, and COVID-19 ARDS, CA), neutrophil isolation, and workflow depicted. ( B ) Representative side scatter (SSC) vs. forward scatter (FSC) plots of stained whole blood from HC, NA and CA displaying lymphocyte (green), monocyte (pink), mature (CD16+, orange) and immature (CD16-, blue) neutrophil populations. ( C ) Representative CD16 vs. CD10 dot plots of stained polymorphonuclear (PMN) and peripheral blood mononuclear cell (PBMC) layers isolated by Percoll gradients from HC (grey), NA (blue) or CA (pink) patients, with quadrant2 (Q2) delimiting the CD16+CD10+ (mature neutrophils) area. ( D - F ) Surface expression of neutrophil activation markers expressed as a fold change of geometric mean fluorescence intensity (MFI) from normal density neutrophils (NDN) respective to the disease state as determined by flow cytometry analysis of mature NDN (CD66b+CD16+, open bars), mature low density neutrophils (LDN) (CD66b+CD16+, horizontal striped bars) and immature LDN (CD66b+CD16-, vertical striped bars) from NA (n = 5), MC (n = 3), or COVID-19 (n = 11; open circles, dexamethasone treated patients) patients. Data are mean +- SD. *p < 0.05, determined by repeated two-way ANOVA and Sidak's post hoc-testing. ( G ) Total neutrophil counts of HC (n = 8), NA (n = 5), MC (n=3) and CA (n = 11) performed by haem
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Experimental details: Figure 6 Effects of Lnc-S100B-2 and MLLT10 on the tumor microenvironment. (a) The expressions of CD3 were detected by IF assay. (b) The percent of CD3 (-) CD16 (+) cells was detected by flow cytometry. (c) The percent of CD11b (+) cells was analyzed by flow cytometry. (d, e) The expression E-cadherin and vimentin was analyzed by IF assay. (f-l) The expressions of E-cadherin, N-cadherin, vimentin, beta -catenin, snail, and slug were detected by qRT-PCR and Western blot. * P < 0.05 versus NC group, # P < 0.05 versus sh-MLLT10 group, & P < 0.05 versus sh-Lnc-S100B-2 group, and one-way ANOVA.
Conjugate: Green dye

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Experimental details: Figure 4 Flow cytometry analysis of PBMCs Flow cytometry gating strategy to analyze PBMCs. CD14 and CD16 were used to distinguish low-density neutrophils (LDNs), non-classical monocytes, intermediate monocytes, and classical monocytes. Gray = negative control; Red = labeled sample.
Conjugate: Green dye

Supportive validation

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Experimental details: Figure 2 IL-1beta-treated primary synoviocytes affected M1/M2 polarization. The primary synoviocytes were treated with IL-1beta (10 ng/mL) for 24 h and then replaced with fresh medium; 24 h later, the CM was collected to treat THP-1 cells. ( a ) Quantitative PCR analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( b ) ELISA analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( c , d ) Flow cytometry analysis of the expression of CD206, a marker of M2, and the expression of CD16, a marker of M1. Data are expressed as the mean +- SD, * p < 0.05. IgG as a negative control. The data presented are representative of three independent experiments.
Conjugate: Green dye

Supportive validation

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Experimental details: Figure 4 Knockdown GRP78 in synoviocytes regulated macrophage polarization to promote the M2 phenotype, using knockdown GRP78 in IL-1beta (10 ng/mL) treated synoviocytes. Then, the CM was collected from synoviocytes to treat THP-1 cells. ( a ) Quantitative PCR analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( b ) ELISA analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( c , d ) Flow cytometry analysis of the expression of CD206, a marker of M2, and the expression of CD16, a marker of M1. Data are expressed as mean +- SD, * p < 0.05. IgG as a negative control. The data presented are representative of three independent experiments.
Conjugate: Green dye

Supportive validation

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Experimental details: Figure 6 HMW-HA treatment in synoviocytes affected macrophage polarization toward the M2 phenotype. HMW-HA treated in knockdown GRP78 synoviocytes, and the supernatant was then collected from synoviocytes as CM and then used to treat THP-1. ( a ) Quantitative PCR analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( b ) ELISA analysis of IL-8, IL-10, IL-12, and TNF-alpha. ( c , d ) Flow cytometry analysis of the expression of CD206, a marker of M2, and the expression of CD16, a marker of M1. Data are expressed as a mean +- SD, * p < 0.05. IgG as a negative control. The data presented are representative of three independent experiments.
Conjugate: Green dye

Supportive validation

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Experimental details: Figure 3 Retinoic Acid Inhibition Increases the Differentiation Capacity of Hematopoietic Progenitors toward Myeloid and Lymphoid Lineage (A) CFU potential of 500 sorted progenitors (CD45/CD43 + CD34 + ) from DEAB-treated cultures (green) and DMSO control (gray) from four independent experiments (n = 4). CFU potential of progenitors similarly sorted from cord blood (CD45/CD43 + CD34 + ) is shown in red. (B) Lymphoid/myeloid differentiation assay performed on iPSC-derived hematopoietic cells. FACS plots and bar graphs showing non-myeloid (CD33/CD14 - ), CD19 - , CD56 + , and CD56 + CD16 + NK fraction from wells seeded with 50 HSC-like cells (CD45/43 + CD34 hi CD38 lo CD90 + CD45RA - ) (17 individual wells) cultured on OP9 stroma for 4 weeks. Left bar graph shows the cluster size of the NK fraction. Right bar graph shows the frequency of this population in the viable non-stroma fraction. See also Figure S2 . (C) FACS plots showing non-myeloid, CD7 + , CD45RA + multipotent early lymphoid progenitors from 3,500 sorted CD34 + cells cultured on OP9DL1 stroma for 3 weeks from one out of two independent experiments (n = 2). FACS plots are gated on the viable non-stroma fraction. Bar graph show the total output of differentiated cells from progenitors generated with DMSO, DEAB, or RA. FACS gates are based on FMO controls. Data represent mean +- SEM. Asterisks indicate significant differences ( * p < 0.05).
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Supportive validation

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Experimental details: Figure 3 Construction of the PVAX1-HPV58 mE6E7FcGB plasmid and expression of the target proteins assessed by immunofluorescence staining. Notes: ( A ) Diagram of the construction of the recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB. HPV58 mE6E7 was cloned into the plasmid PCI-sig-Fc-GPI, and the fused cassette sig-HPV58 mE6E7-Fc-GPI was subsequently excised from PCI-sig-HPV58 mE6E7-Fc-GPI plasmid and inserted into the upstream of IRES in PVAX1-IRES-GM/B7.1 vector to create PVAX1-HPV58 mE6E7FcGB. ( B ) The transfected 293T cells were concurrently incubated with a rabbit anti-human IgG-FITC antibody (green) and a mouse anti-human B7-1-PE antibody (red). Immunofluorescence revealed the expression of sig-HPV58 mE6E7-Fc-GPI (a, green, x50) and GM-CSF/B7.1 (b, red, x50) at the cells membrane. Abbreviations: IgG-FITC, immunoglobulin G-conjugated fluorescein isothiocyanate; IRES, internal ribosome entry site; HPV, human papillomavirus.
Conjugate: Green dye

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Experimental details: Fig. 3 HIV-specific responses and immune response capability of the case at 9.5 years of age. a Detection of HIV-specific antibodies at 9.5 years of age by western blot. The case antibody profile is compared with controls that are a high positive, low positive and HIV-negative. HIV proteins corresponding to bands in the blots are shown in the left grey-shaded block; the case profile was positive for the core proteins indicated in pink. b Quantitation of HIV-specific antibodies by multiplex bead array for all isotypes and subclasses (indicated on the left side--IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2) against gp41, Gag, RT, gp120, Nef, Tat, Vpu, peptide Vpu9 and V1V2 scaffold antigens (indicated at the top). Results are expressed as mean fluorescence intensities (MFI)), the colour key shows ranges of MFI according to colour intensity (the darker the more HIV-specific antibody detected). A result is considered positive if above the cut-off (mean +- 3 SD) determined from eight adult uninfected controls. Vpu9 amino acid sequence: STMVDMGHLRLLDVNDL. c Proportions of natural killer (NK) cells that respond to anti-CD16 antibody, and CD4+ and CD8+ T cells that respond to staphylococcal enterotoxin B (SEB) in a whole blood intracellular cytokine (ICC) assay that measures induction of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). HIV-uninfected adult reference values for comparison ( n = 21; median % and range)--natural killer (NK) anti-CD16%: 37.92 (12-67.6), CD4 SEB%:
Conjugate: Green dye

Supportive validation

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Experimental details: Fig. 5 Analysis of neutrophils from control or androgen deprivation therapy (ADT) treated patients. a Percent of circulating neutrophils from control or ADT treated prostate cancer patients that have a mature, segmented appearance morphologically. b Percent Cd11b + CD66b + CD16 lo neutrophils. c Percent myeloid derived suppressor cell (MDSC)-like neutrophils defined as HLA-DR - CD14 - CD33 + CD15 + . d Percent fold change in migration (percent crystal violet positive area of transwell stimulated with a chemoattractant divided by percent area without chemoattractant). e Percent of unstimulated neutrophils producing DHR123. f Percent of neutrophils producing DHR123 following PMA stimulation. g Percent myeloperoxidase + (MPO + ) neutrophils. h Percent of plated netosising neutrophils at 1 and 3 h following purification. Data in a - h are mean +- s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 by multivariable linear regression using ADT as its main predictor and adjusting for presence of prostatectomy, age, race, and current status; control prostate cancer patients ( n = 8 in a , c , g and n = 7 in b , d , e , f , h ) and prostate cancer pati e nts receiving ADT ( n = 22 in a , c , g and n = 21 in b , d , e , f , h ).
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Supportive validation

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Experimental details: Fig. 1 Gating strategy for flow cytometry sorting of human CD14 + CD16 + and CD14 + CD16 - monocytes. Peripheral blood mononuclear cells from RA and HC were stained with FITC-conjugated anti-CD14 antibody and APC-conjugated anti-CD16 antibody. CD14 + CD16 + and CD14 + CD16 - monocytes were analyzed and sorted by flow cytometric; the purity of sorted CD14 + CD16 + and CD14 + CD16 - monocytes used for experiments was ~ 90%
Conjugate: Green dye

Supportive validation

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Experimental details: Fig. 2 CD14 + CD16 - monocytes are the main osteoclast precursors in RA. Purified CD14 + CD16 + and CD14 + CD16 - monocytes from RA ( n = 5) and HC ( n = 5) were cultured with rhM-CSF (30 ng/ml) and rhRANKL (50 ng/ml) for osteoclast differentiation. The cells were detected for tartrate-resistant acid phosphatase (TRAP) staining on day 17, and the TRAP-positive multinuclear cells were osteoclasts. The representative charts and the statistical results were shown. a CD14 + CD16 + versus CD14 + CD16 - monocytes in HC (* P = 0.026). b RA versus HC for CD14 + CD16 - monocytes (* P = 0.019). c RA versus HC for CD14 + CD16 + monocytes. * P < 0.05; ns, not significant (Student''s t test, a - c )
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Supportive validation

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Experimental details: Fig. 3 The expression of Tyro3TK on CD14 + CD16 - monocytes is increased in RA. a Gating strategy for identifying the expression of Tyro3TK on CD14 + CD16 + and CD14 + CD16 - monocytes. Accordingly, the expression of Tyro3TK on CD14 + CD16 + and CD14 + CD16 - monocytes in HC ( n = 40) ( b ), OA ( n = 28) ( c ), and RA patients ( n = 40, ** P = 0.008) ( d ) were analyzed and presented as the mean fluorescence intensity (MFI). e The expression of Tyro3TK on CD14 + CD16 + monocytes were compared between HC, OA, and RA patients. f The expression of Tyro3TK on CD14 + CD16 - monocytes were compared between HC, OA, and RA patients (** P = 0.004, *** P < 0.001). g Flow cytometry-sorted CD14 + CD16 - monocytes from RA ( n = 4) and HC ( n = 4) were set to detect the mRNA expression of Tyro3TK by qPCR (* P = 0.029). * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant (Mann-Whitney U test, b , d , and g ; Student's t test, c ; Kruskal-Wallis test followed by Dunn's post-test for multiple comparisons, e - f )
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Supportive validation

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Experimental details: Fig. 4 Correlation analysis of Tyro3TK on CD14 + CD16 - monocytes with RA patient clinical manifestations. The associations of Tyro3TK on CD14 + CD16 - monocytes with RA patient DAS28-ESR ( r = 0.323, * P = 0.042) ( a ), tender joint counts (TJC) ( r = 0.514, ** P = 0.001) ( b ), and IgM ( r = 0.432, ** P = 0.005) ( c ) were analyzed. The expression of Tyro3TK on CD14 + CD16 - monocytes were also compared between the different RA patient groups: d RA with high disease activity (DAS28-ESR > 5.1) and non-high disease activity (DAS28-ESR
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Supportive validation

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Experimental details: Fig. 5 Tyro3TK promotes CD14 + CD16 - monocyte-mediated osteoclastogenesis in RA. Purified CD14 + CD16 - monocytes from RA patients ( n = 8) were cultured with rhM-CSF (30 ng/ml) and rhRANKL (50 ng/ml) under different conditions for osteoclast differentiation. Seventeen days later, the cells were harvested for TRAP staining. The representative charts and the statistical results were shown. a Different concentrations of rhGas6 (0 ng/ml, 10 ng/ml, 50 ng/ml, and 100 ng/ml) were supplemented for osteoclast differentiation ( n = 3 per group, ** P = 0.006). b Different concentrations of anti-Tyro3TK antibody (0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml), and 50 ng/ml rhGas6 was supplemented for osteoclast differentiation ( n = 5 per group, *** P < 0.001). ** P < 0.01, *** P < 0.001 (one-way ANOVA test followed by Dunn''s post-test for multiple comparisons, a , b )
Conjugate: Green dye