Anti-CD3 epsilon antibody [SP7] (ab16669)
Key features and details
- Rabbit monoclonal [SP7] to CD3 epsilon
- Suitable for: Flow Cyt (Intra), IHC-P, WB, mIHC
- Reacts with: Mouse, Rat, Human
- Isotype: IgG
Related conjugates and formulations
Overview
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Product name
Anti-CD3 epsilon antibody [SP7]
See all CD3 epsilon primary antibodies -
Description
Rabbit monoclonal [SP7] to CD3 epsilon -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), IHC-P, WB, mIHCmore details -
Species reactivity
Reacts with: Mouse, Rat, Human
Predicted to work with: Sheep, Rabbit, Horse, Chicken, Cow, Cat, Dog, Pig, Baboon, Woodchuck -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Recombinant Human CD3 epsilon protein (ab114153), Jurkat whole cell lysate. Human, mouse and rat thymus tissue lysate. IHC-P: Human tonsil tissue. Mouse epidydimal fat pad and lymph node tissues. Rat infarcted heart and spleen tissues. Flow Cyt (intra): Human peripheral blood lymphocytes. Jurkat cells. mIHC: Human tonsil, duodenum, and colon tissue. Rat and mouse spleen tissue.
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General notes
We recommend ab135372 as an alternative.
This antibody is suitable for staining normal and neoplastic T cells in formalin-fixed, paraffin-embedded tissues.
Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077). Or search our wide range of secondary antibodies for use with your experiment.This product is FOR RESEARCH USE ONLY. For commercial use, please contact partnerships@abcam.com.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid repeated freeze / thaw cycles. -
Storage buffer
pH: 7.50
Preservative: 0.1% Sodium azide
Constituents: Tissue culture supernatant, Tris buffered saline, 1% BSA -
Concentration information loading...
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Purity
Tissue culture supernatant -
Primary antibody notes
This antibody is suitable for staining normal and neoplastic T cells in formalin-fixed, paraffin-embedded tissues. -
Clonality
Monoclonal -
Clone number
SP7 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab16669 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) | ||
IHC-P | (57) |
1/150. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min. |
WB | (1) |
1/25. Predicted molecular weight: 23 kDa.
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mIHC |
1/500.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) |
Notes |
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IHC-P
1/150. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min. |
WB
1/25. Predicted molecular weight: 23 kDa. |
mIHC
1/500. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) |
Target
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Function
The CD3 complex mediates signal transduction. -
Sequence similarities
Contains 1 Ig-like (immunoglobulin-like) domain.
Contains 1 ITAM domain. -
Cellular localization
Membrane. - Information by UniProt
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Database links
- Entrez Gene: 281054 Cow
- Entrez Gene: 442981 Dog
- Entrez Gene: 916 Human
- Entrez Gene: 12501 Mouse
- Entrez Gene: 397455 Pig
- Entrez Gene: 100008616 Rabbit
- Entrez Gene: 315609 Rat
- Omim: 186830 Human
see all -
Alternative names
- CD3 epsilon antibody
- CD3e antibody
- CD3e antigen antibody
see all
Images
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Immunohistochemical analysis of formalin fixed paraffin (FFPE) embedded tonsil labelling CD3 with ab16669 at a dilution of 1/600. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an ChromoMap DAB (RUO) IHC Detection Kit with anti rabbit HQ and anti HQ HRP. Heat mediated antigen retrieval was conducted for 24 min with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5. ab16669 was incubated at 37°C for 16 min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
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Chromogenic multiplex immunohistochemical staining of FFPE normal human tonsil tissue. Ab16667, anti-Ki67 DAB chromogen. Ab16669, anti-CD3 purple chromogen and ab192847, anti-CD68 teal chromogen plus haematoxylin II counterstain.
Chromogenic immunostaining was performed on a Roche Ventana Benchmark Ultra instrument. The section was deparaffinised and incubated with CC1 solution for 24min, 100°C. Following this, with 3 rounds of staining in the order of ab16667 (1/500), ab192847 (1/4000) ab16669 (1/1000). Between rounds of staining, antibody denaturation was conducted using Ultra CC2 solution for 8min at 100°C to avoid cross reactivity. Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with Chromomap DAB kit, Discovery purple or Discovery teal chromogens and haematoxylin II counterstain. -
Panel A: merged staining of anti-CD68 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, and couterstaining was with DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilutionThe section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
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Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, and couterstaining was with DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
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Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells and immune cell subsets with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins -
Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
Panel B: anti-CD3 stained on T cells with ab16669 at 1/500 dilution
Panel C: anti-CD19 stained on B cells with ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins -
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human thymus tissue labeling CD3 with ab16669 at 1/500 dilution, LY75/DEC-205 with ab208649 at 1/15000, and CD68 with ab213363 at 1/500 dilution.
Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 (green; Opal™520) and anti-LY75/DEC-205 (red; Opal™570) on human thymus.
Panel B: anti-CD3 stained on T cells.
Panel C: anti-LY75/DEC-205 stained on thymic cortical epithelium and dendritic cells.
Panel D: anti-CD68 stained on macrophages.
Sections were treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins before antibody incubation. The section was incubated in three rounds of staining: in the order of ab213363, ab16669, and ab208649 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.DAPI was used as a nuclear counterstain.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. -
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat spleen tissue labeling Sialoadhesin/CD169, CD3 and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and ab236434 at 1:5000 dilution.
Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 (green; Opal™520) and anti-CD20 (gray; Opal™570) on rat spleen.
Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
Panel C: anti-CD3 stained on T cells.
Panel D: anti-CD20 stained on B cells.The section was incubated in three rounds of staining: in the order of ab312840, ab16669, and ab236434 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
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Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen tissue labeling Sialoadhesin/CD169, CD3 and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and ab236434 at 1:5000 dilution.
Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 (green; Opal™520) and anti-CD20 (gray; Opal™570) on mouse spleen.
Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
Panel C: anti-CD3 stained on T cells.
Panel D: anti-CD20 stained on B cells.The section was incubated in three rounds of staining: in the order of ab312840, ab16669, and ab236434 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
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Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 (ab16669, green; Opal™520) and anti-CD68 (ab213363, red; Opal™570) on human colon. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and ab213363 (1/500 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
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Fluorescence multiplex immunohistochemical analysis of the human duodenum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 (ab16669, green; Opal™520) and anti-CD68 (ab213363, red; Opal™570) on human duodenum. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and ab213363 (1/500 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.
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IHC image of CD3 staining in a formalin fixed, paraffin embedded normal rat spleen tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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All lanes : Anti-CD3 epsilon antibody [SP7] (ab16669) at 1/25 dilution
Lane 1 : THP1 whole cell lysate (-ve control)
Lane 2 : Raji whole cell lysate (-ve control)
Lane 3 : Jurkat whole cell lysate
Lane 4 : Human Thymus tissue lysate
Lane 5 : Mouse Thymus tissue lysate
Lane 6 : Rat Thymus tissue lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDaLanes 1 - 6: Merged signal (red and green). Green – ab16669 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16669 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.
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Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).
Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®).
The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.
Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.
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Human peripheral blood lymphocytes stained with ab16669 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab16669, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.
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IHC image of CD3 staining in a formalin fixed, paraffin embedded normal human tonsil tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
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IHC image of CD3 staining in mouse lymph node formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times
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Immunohistochemical analysis of Formaldehyde fixed, paraffin-embedded rat spleen tissue sections labelling CD3 with ab16669 at a dilution of 1/100. Biotin conjugated Goat Anti-Rabbit IgG at 1/300 dilution was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid.
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IHC using multi-spectral imaging on human lymph node (A-C) obtained from men with mCRPC. A) H+E staining and B) single-color images (plus nuclear stain; DAPI) of CD3 (ab16669), CD8 (ab101500), CD163, PD-L1, cytokeratin (CK), DAPI and C) merged. H+E stating at 20X magnification; multi-spectral images 200X magnification.
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Ab16669 staining CD3 in Mouse Epidydimal fat pad tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue sections were fixed with formaldehyde, blocked with 5% serum for 4 hours at 25°C and permeabilized with Triton X-100. Samples were incubated with primary antibody (1/100 in PBST with BSA and goat serum) for 4°C at 12 hours. An Alexa Fluor® 568 goat anti-rabbit IgG (H + L) cross adsorbed was used as the secondary antibody.
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Flow cytometric analysis of rabbit anti-CD3 (SP7) antibody ab16669 (1/100) in Jurkats cells (green) compare to negative control of rabbit IgG (blue).
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ab16669 staining rat infarcted heart tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
Myocardial infarction was produced in a rat model following the ligation of the left anterior descending (LAD) coronary artery. Tissue was harvested 6 w following infarct, fixed with Histochoice for 72 hr, paraffin sectioned and the slide was then baked prior to CD3 staining. ab16669 at 1/200 was incubated overnight at 4°C. The image was taken with a confocal laser scanning microscope and shows cells giving strong inmmunofluorescence staining for CD3 antigen (green), indicating presence of cells of T-lymphocytes origin in the infarct zone of the heart tissue, counterstained nuclei with DAPI (blue). Note, CD3 tended to be present in nests of 2-5 cells that were non-uniformly distributed in the infarct zone. In addition, the image shows that the CD3 localization is predominantly membrane based and to a certain extent intracytoplasmic.
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Immunohistochemical analysis of Human tonsil tissue, staining CD3 (green) with ab16669.
Antigen retrieval was performed by heat mediation in citrate buffer (pH 6) and blocked with 5% goat serum and 5% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/100) overnight at 4°C. A Cy3®-conjugated anti-rabbit IgG was used as the secondary antibody.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (612)
ab16669 has been referenced in 612 publications.
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