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A guide to selecting high-performing antibodies for TGF-beta receptor type-2 (UniProt ID: P37173) for use in western blot and flow cytometry [version 1; peer review: awaiting peer review]
Дата публикации: 12-08-2026 06:09:19
Idiopathic pulmonary fibrosis is a chronic, progressive interstitial lung disease characterised by excessive extracellular matrix deposition, leading to irreversible lung remodelling and respiratory failure. Transforming growth factor-beta 1 is a central driver of fibrogenesis, and its binding to transforming growth factor-beta receptor type 2 (TGFBR2) is essential for activation of both canonical and non-canonical signalling pathways. Given its critical role, TGFBR2 represents an important target for investigating disease pathogenesis and developing therapeutic strategies. However, the performance and specificity of commercially available antibodies targeting TGFBR2 have not been systematically evaluated, limiting reproducibility and confidence in experimental findings. In this study, we systematically assessed eleven commercial antibodies by western blot and twelve by flow cytometry using a standardised knockout-based validation strategy in human A549 cells. Antibody performance was evaluated by comparing signals in TGFBR2 knockout cells with isogenic parental controls. These experiments are part of a broader collaborative effort to improve antibody reproducibility through the systematic characterisation of commercial reagents and open dissemination of results. Although antibody performance may vary depending on experimental conditions, this work provides a practical resource to guide the selection of appropriate antibodies for studying TGFBR2 in health and disease.
Основное содержимое страницы с новостью.
Biddle M, Cooper J, Jones C et al. A guide to selecting high-performing antibodies for TGF-beta receptor type-2 (UniProt ID: P37173) for use in western blot and flow cytometry [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:1355 (https://doi.org/10.12688/f1000research.185842.1)
Data Note
[version 1; peer review: awaiting peer review]
https://orcid.org/0000-0002-9853-8815
1, Jemma Cooperhttps://orcid.org/0009-0007-8915-379X
1, Carolyn Jones1, Katie Dixonhttps://orcid.org/0009-0007-1014-0866
1, Harvinder Virkhttps://orcid.org/0000-0002-9739-9593
1https://orcid.org/0000-0002-9853-8815
1, Jemma Cooperhttps://orcid.org/0009-0007-8915-379X
1, [...] Carolyn Jones1, Katie Dixonhttps://orcid.org/0009-0007-1014-0866
1, Harvinder Virkhttps://orcid.org/0000-0002-9739-9593
11 University of Leicester College of Life Sciences, Leicester, England, UK
Michael Biddle
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing
Jemma Cooper
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Review & Editing
Carolyn Jones
Roles: Investigation, Methodology, Project Administration
Katie Dixon
Roles: Data Curation, Writing – Original Draft Preparation, Writing – Review & Editing
Harvinder Virk
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing
OPEN PEER REVIEW
REVIEWER STATUS AWAITING PEER REVIEW
Abstract
Idiopathic pulmonary fibrosis is a chronic, progressive interstitial lung disease characterised by excessive extracellular matrix deposition, leading to irreversible lung remodelling and respiratory failure. Transforming growth factor-beta 1 is a central driver of fibrogenesis, and its binding to transforming growth factor-beta receptor type 2 (TGFBR2) is essential for activation of both canonical and non-canonical signalling pathways. Given its critical role, TGFBR2 represents an important target for investigating disease pathogenesis and developing therapeutic strategies. However, the performance and specificity of commercially available antibodies targeting TGFBR2 have not been systematically evaluated, limiting reproducibility and confidence in experimental findings.
In this study, we systematically assessed eleven commercial antibodies by western blot and twelve by flow cytometry using a standardised knockout-based validation strategy in human A549 cells. Antibody performance was evaluated by comparing signals in TGFBR2 knockout cells with isogenic parental controls. These experiments are part of a broader collaborative effort to improve antibody reproducibility through the systematic characterisation of commercial reagents and open dissemination of results. Although antibody performance may vary depending on experimental conditions, this work provides a practical resource to guide the selection of appropriate antibodies for studying TGFBR2 in health and disease.
Keywords
P37173, TGFBR2, TGF-beta receptor type-2, western blot, flow cytometry, idiopathic pulmonary fibrosis, IPF
Corresponding authors: Michael Biddle, Harvinder Virk Competing interests: Michael Biddle and Harvinder Virk have received funding for a research studentship from Abcam Ltd, as well as in-kind contributions from manufacturers that contribute to the YCharOS Inc. consortium. The remaining authors declare no competing interests. These relationships did not influence the study design, data collection or analysis, decision to publish, or preparation of the manuscript.
Grant information: This work was supported by a grant from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and MRC (NC3Rs Ref: NC/NAM0019/1, MRC UKRI076) alongside support from the Leicester Institute for Precision Health.
The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright: © 2026 Biddle M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Biddle M, Cooper J, Jones C et al. A guide to selecting high-performing antibodies for TGF-beta receptor type-2 (UniProt ID: P37173) for use in western blot and flow cytometry [version 1; peer review: awaiting peer review]. F1000Research 2026, 15:1355 (https://doi.org/10.12688/f1000research.185842.1) First published: 12 Aug 2026, 15:1355 (https://doi.org/10.12688/f1000research.185842.1) Latest published: 12 Aug 2026, 15:1355 (https://doi.org/10.12688/f1000research.185842.1)
Introduction
Idiopathic pulmonary fibrosis is a chronic, progressive interstitial lung disease, characterised by excessive deposition of extracellular matrix proteins, leading to irreversible architectural distortion of the lung and ultimately respiratory failure.1 Among the key drivers of fibrogenesis, transforming growth factor-beta 1 (TGFB1) is widely recognised as a central mediator, promoting fibroblast activation, myofibroblast differentiation, and extracellular matrix production.2 To initiate this signalling cascade, TGFB1 binds to transforming growth factor-beta receptor type 2 (TGFBR2), which then recruits and phosphorylates transforming growth factor-beta receptor type 1 (TGFBR1), inducing SMAD2/3 phosphorylation.3
As such, TGFBR2 represents an important target for understanding the molecular mechanisms underlying IPF and for the development of potential therapeutic strategies. However, the reliability of commercially available antibodies targeting TGFBR2 has not been systematically evaluated, limiting the reproducibility and interpretability of experimental findings. Robust and well-validated antibodies are therefore essential to accurately detect TGFBR2 expression, localisation, and regulation across experimental systems.
This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterising commercial antibodies for human proteins using standardised protocols4 and openly sharing the data.5 Here we evaluated the performance of eleven commercial antibodies for TGFBR2 by western blot and twelve commercial antibodies by flow cytometry, enabling biochemical and cellular assessment of TGFBR2 properties and function. The platform for antibody characterisation used to carry out this study was endorsed by a committee of industry and academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterisation procedures using most commercially available renewable antibodies against the corresponding protein. The standardised consensus antibody characterisation protocols are openly available on Protocols.io (DOI: dx.doi.org/10.21203/rs.3.pex-2607/v1).
The authors do not engage in result analysis or offer explicit antibody recommendations. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterisation data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret the antibody characterisation data found in this study are featured on the YCharOS gateway.6
Results and discussion
Our standard protocol involves comparing readouts from WT (wild type) and KO cells.7–9 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcript per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). The A549 cell line expresses the TGFBR2 transcript at 5.6 log2 TPM+1, and a TGFBR2 KO A549 cell line was obtained from Abcam ( Table 1).
Table 1. Summary of the cell lines used.InstitutionCatalogue numberRRID (Cellosaurus)Cell lineGenotypeAbcamAB288558CVCL_0023A549WTAbcamAB261863CVCL_B2QIA549TGFBR2 KO
To screen TGFBR2 antibodies by western blot, protein lysates from both A549 WT and TGFBR2 KO cell lines were run on SDS-PAGE, transferred onto nitrocellulose membranes and probed in parallel with eleven TGFBR2 antibodies ( Figure 1).
Figure 1. TGFBR2 antibody screening by western blot using protein lysates.
Protein lysates from A549 WT and TGFBR2 KO cells were collected, and 30μg of protein was used for western blot with the indicated TGFBR2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO samples. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilutions used: ab184948** at 1/1000, ab259360** at 1/1000, ab270440** at 1/500, ARP44743_T100 at 1/500, 41896** at 1/1000, GTX129909 at 1/1000, 66636-1-Ig* at 1/1000, MAB2411* at 1/500, 701683** at 1/500, MA5-51441** at 1/1000, MA5-55343** at 1/1000. Predicted band size: 64.6kDa. ** = recombinant antibody, * = monoclonal antibody.
For flow cytometry, A549 WT and TGFBR2 KO cells were labelled with distinct fluorescent dyes and combined at a 1:1 ratio. Both cell lines were fixed, permeabilised and blocked in the same tube prior to antibody staining to reduce bias. Twelve TGFBR2 antibodies were then evaluated, with fluorescence intensity assessed using the Attune NxT flow cytometer. Antibody staining in both the WT and KO lines was then quantified using FlowJo software, with representative histograms presented in Figure 2.
To assess cell surface labelling, a separate 1:1 mix of dye-labelled A549 WT and TGFBR2 KO cells was prepared without fixation or permeabilisation, such that only surface-accessible epitopes were detected. Both cell lines were blocked in the same tube, then stained with the same twelve TGFBR2 antibodies before gating out dead cells with sytox orange. Fluorescence intensity was then acquired on the Attune NxT flow cytometer and quantified in both the WT and KO lines using FlowJo software, with representative histograms presented in Figure 3.
Figure 2. TGFBR2 antibody screening by flow cytometry: intracellular staining.
A549 WT and TGFBR2 KO cells were labelled with a green or violet fluorescent dye, respectively. WT and KO cells were mixed in a 1:1 ratio, fixed in 4% PFA and permeabilised in 0.1% saponin. 400,000 cells were stained with the indicated TGFBR2 antibodies and corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies (except 130-115-067 which was conjugated). Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the KO population (pink histogram, dashed line) compared to the WT cells (green histogram, solid line). For unconjugated antibodies grey histograms with dotted lines represent secondary antibody-only controls in both WT and KO cells. For conjugated antibodies blue histograms with dotted lines represent fluorescence minus one control in both WT and KO cells. ab184948** at 1/2326, ab259360** at 1/469, ab270440** at 1/584, ARP44743_T100 at 1/1000, 41896** at 1/340, GTX129909 at 1/360, 130-115-067** at 1/1000, 66636-1-Ig* at 1/1000, MAB2411* at 1/500, 701683** at 1/500, MA5-51441** at 1/203, MA5-55343** at 1/2000. ** = recombinant antibody, * = monoclonal antibody.
Figure 3. TGFBR2 antibody screening by flow cytometry: cell surface.
A549 WT and TGFBR2 KO cells were labelled with a green or violet fluorescent dye, respectively. WT and KO cells were mixed in a 1:1 ratio, and 400,000 cells were stained with the indicated TGFBR2 antibodies. Multi-rAb CoraLite® Plus 647 secondary antibodies were then added, except for 130-115-067 which was conjugated. Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the KO population (pink histogram, dashed line) compared to the WT cells (green histogram, solid line). For unconjugated antibodies grey histograms with dotted lines represent secondary antibody-only controls in both WT and KO cells. For conjugated antibodies blue histograms with dotted lines represent fluorescence minus one control in both WT and KO cells. ab184948** at 1/2326, ab259360** at 1/469, ab270440** at 1/584, ARP44743_T100 at 1/1000, 41896** at 1/340, GTX129909 at 1/360, 130-115-067** at 1/100, 66636-1-Ig* at 1/1000, MAB2411* at 1/500, 701683** at 1/500, MA5-51441** at 1/203, MA5-55343** at 1/2000. ** = recombinant antibody, * = monoclonal antibody.
In conclusion, we screened eleven commercial TGFBR2 antibodies by western blot and twelve by flow cytometry, comparing signals obtained from human A549 wild-type and TGFBR2 knockout cells. High-quality, renewable antibodies capable of reliably detecting TGFBR2 were identified.
Limitations
Inherent limitations are associated with the antibody characterisation platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available TGFBR2 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. We encourage readers to consult vendor documentation to identify the specific antigen each antibody is raised against, where such information is available.
Secondly, the YCharOS effort employs an unbiased approach that is agnostic to the protein for which antibodies have been characterised. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in TGFBR2, only a brief overview of the protein's function and its relevance in disease is provided. TGFBR2 experts are invited to analyse and interpret observed banding patterns in western blots. Thirdly, YCharOS experiments are not performed in replicate primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent characterisation is performed subsequent to the antibody manufacturer’s internal validation process, therefore making our characterisation process a repeat.
Lastly, as comprehensive and standardised procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.
Methods
The standardised protocols used to carry out this KO cell line-based antibody characterisation platform were established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterise antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocols.io (DOI: dx.doi.org/10.21203/rs.3.pex-2607/v1).
Antibodies
All TGFBR2 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure antibodies are cited properly.10 Secondary antibodies used in this study are provided in Table 3. To ensure consistency with manufacturer recommendations and account for proprietary formulations (where antibody concentrations are not disclosed), antibody usage is reported as dilution ratios rather than absolute concentrations.
Table 2. Summary of the TGFBR2 antibodies tested.CompanyCatalogue numberLot numberRRID (Antibody Registry)ClonalityClone IDHostStock concentration (μg/mL)Vendor recommended applicationsAbcamab184948**1062725-6AB_2818975Monoclonal RecombinantEPR14673Rabbit2326WBAbcamab259360**1001702-24AB_3668832Monoclonal RecombinantEPR24349-124Rabbit469IP, WBAbcamab270440**1025845-8AB_3741053Monoclonal RecombinantEPR23237-2Rabbit584FC, ICC/IFAviva Systems BiologyARP44743_T100QC14853-20071108AB_938452Polyclonal-Rabbit1000WBCell Signalling Technology41896**2AB_3668871Monoclonal RecombinantE5M6FRabbit340WBGeneTexGTX12990941836AB_2886121Polyclonal-Rabbit360WBMiltenyi Biotec130-115-067**5250201912AB_2726890Monoclonal RecombinantREA903Human-FC-CS, ICC/IF, IHCProteinTech66636-1-Ig*10023257AB_2881995Monoclonal2D5H7Mouse1000ELISA, IHC, WBR & D SystemsMAB2411*ITL0324051AB_2202474Monoclonal176510Mouse500WBThermo Fisher Scientific701683**2827651AB_2608868Monoclonal Recombinant16H2L4Rabbit500ICC/IF, WBThermo Fisher ScientificMA5-51441**ZJ4521736AAB_3093813Monoclonal Recombinant8A3Q9Rabbit203ELISA, IHC, WBThermo Fisher ScientificMA5-55343**ZJ4521730AB_3667584Monoclonal Recombinant2M8Y2Rabbit2000ELISA, WB
Table 3. Summary of the secondary antibodies used.CompanySecondary antibodyCatalogue numberRRID (Antibody Registry)ClonalityApplicationStock concentration (μg/mL)Working concentration (μg/mL)ProteintechHRP-Goat Anti-Rabbit (H+L)RGAR001AB_3073505Recombinant PolyclonalWestern blot10000.1ProteintechHRP-Goat Anti-Mouse (H+L)RGAM001AB_3068333Recombinant PolyclonalWestern blot10000.1ProteintechCoralite-Plus-647-Goat Anti-Rabbit (H + L)RGAR005AB_3073509Recombinant PolyclonalFlow cytometry5000.625ProteintechCoralite-Plus-647-Goat Anti-Mouse (H + L)RGAM005AB_3073503Recombinant PolyclonalFlow cytometry5000.625
Cell culture
All cell lines used in this study are listed in Table 1, alongside their corresponding RRIDs, to ensure proper citation.11 Cells were cultured in DMEM (Capricorn Scientific #DMEM-HPSTA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific #A5256801) and 1% antibiotic/antimycotic solution (Capricorn Scientific #AAS-B). All cell lines used in this study were routinely tested for mycoplasma contamination and were confirmed to be mycoplasma-free.
Antibody screening by western blot
For lysate preparation, A549 WT and TGFBR2 KO cells were washed three times in phosphate buffered saline (PBS) (Thermo Fisher Scientific #70011044) and lysed in RIPA buffer containing 1x of protease inhibitor cocktail, sodium orthovanadate and phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology #sc-24948). Lysates were sonicated (40% amplitude for 5 seconds) three times and incubated for 30 minutes on ice prior to centrifugation at 20,000 × g for 1 hour at 4°C.
Protein concentration was confirmed using the Pierce BCA protein assay (Thermo Fisher Scientific #23225) and 30μg of protein was used. Samples were combined with Laemmli sample buffer (Bio-Rad #1610747) containing 2-mercaptoethanol (final concentration 355mM) (Sigma-Aldrich #M7522) before being heated at 65°C for 10 minutes. Samples were then loaded in precast 4-20% WedgeWell Tris-Glycine Plus midi gels (Thermo Fisher Scientific #WTG42020BOX) alongside Prime-Step prestained broad range protein ladder (BioLegend #773302). SDS-PAGE was then performed in SureLock Tandem Midi Gel tanks (Thermo Fisher Scientific #STM1001) and run at 200V for 1 hour with Tris/Glycine/SDS buffer (Bio-Rad #1610772). Proteins were then transferred to 0.2 μm supported nitrocellulose membranes (Cytiva #10600015) using a Criterion blotter with plate electrodes (Bio-Rad #17004070) run at 85V for 45 minutes. Proteins on the blot were then visualised with Ponceau S staining (Thermo Fisher Scientific #161470250) which was scanned to show alongside individual western blots. Blots were blocked with 5% milk for 1 hour except for antibody 41896 which was blocked in 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) (Thermo Fisher Scientific #J77500.K2). Primary antibodies were then incubated overnight at 4°C in 5% milk TBST with gentle shaking. Following three ten-minute washes with TBST, horseradish peroxidase (HRP) conjugated secondary antibodies were incubated at a dilution of 1/10000 (0.1μg/mL) in TBST with 5% milk for 1 hour at room temperature followed by three ten-minute washes with TBST. Membranes were then incubated with either Pierce ECL (Thermo Fisher Scientific #32106) for 1 minute or Clarity Western ECL substrate (Bio-Rad #1705061) for 5 minutes prior to detection with the ImageQuant LAS 4000.
Antibody screening by flow cytometry: intracellular staining
A549 WT and TGFBR2 KO cells were detached, and seven million cells were labelled with CellTracker green or violet fluorescent dyes, respectively (Thermo Fisher Scientific, #C7025 and #C10094). WT and KO cells were centrifuged at 300 × g, for 10 minutes and resuspended in PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, A9647). The two populations were combined at a 1:1 ratio, centrifuged and fixed on ice for 20 minutes using 800 μL of 4% PFA in PBS (Thermo Fisher Scientific #J19943.K2). Following fixation, 1.2 mL of 1% BSA in PBS was added to the tube, vortexed and centrifuged at 600 × g for 15 minutes at 4°C. Cells were then permeabilised in 400 μL PBS with 0.1% saponin (Sigma-Aldrich #558255) for 10 minutes at room temperature, centrifuged at 600 × g for 15 minutes at 4°C and then blocked with 5% goat serum (Sigma-Aldrich #G6767), 1% BSA, 0.1% saponin in PBS for 30 minutes on ice. After the blocking step 400,000 cells were aliquoted into individually labelled tubes, centrifuged at 600 × g for 15 minutes at 4°C and incubated in 150 μL of 1% BSA, 0.1% saponin PBS with primary TGFBR2 antibodies for 30 minutes on ice. 500 μL of 1% BSA, 0.1% saponin PBS was then added to each tube, vortexed and centrifuged at 600 × g for 15 minutes at 4°C. Cells were then incubated with their corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies ( Table 3) in 150 μL of 1% BSA, 0.1% saponin PBS for 30 minutes on ice. For conjugated antibodies the addition of a secondary antibody was excluded and replaced with 150 μL of 1% BSA, 0.1% saponin in PBS during this incubation. 500 μL of 1% BSA, 0.1% saponin PBS was then added to each tube, vortexed and centrifuged at 600 × g for 15 minutes at 4°C.
Tubes were then resuspended in 1 mL of 1% BSA in PBS and data was acquired using the Attune NxT flow cytometer. Data was analysed using FlowJo with the following gates. The cell population was first gated on FSC-A vs SSC-A, within that gate single cells were selected by FSC-A vs FSC-H and then KO and WT cells were isolated by BL1-A vs VL1-A using a quadrant gate. Antibody staining was then quantified in the RL1-A channel and histograms were merged to demonstrate the staining intensity between the two populations compared to their two respective controls (secondary only or fluorescence minus one). The figure was then assembled using Adobe Illustrator 2024.
Antibody screening by flow cytometry: cell surface staining
For cell surface staining A549 WT and TGFBR2 KO cells were detached, and seven million cells were labelled with CellTracker green or violet fluorescent dyes, respectively (Thermo Fisher Scientific, #C7025 and #C10094). WT and KO cells were centrifuged at 400 × g, for 10 minutes and resuspended in PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, A9647). Cell populations were then combined at a 1:1 ratio, centrifuged and blocked on ice for 30 min with 5% goat serum (Sigma-Aldrich #G6767), 1% BSA in PBS. After the blocking step 400,000 cells were aliquoted into individually labelled tubes, centrifuged at 400 × g for 15 minutes at 4°C, and incubated in 150 μL of 1% BSA PBS with primary TGFBR2 antibodies for 30 minutes on ice. 500 μL of 1% BSA PBS was then added to each tube, vortexed gently and centrifuged at 400 × g for 15 minutes at 4°C. Cells were then incubated for 30 minutes on ice with their corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies ( Table 3) in 150 μL of 1% BSA, PBS. For conjugated antibodies the addition of a secondary antibody was excluded and replaced with 150 μL of 1% BSA PBS during this incubation. 500 μL of 1% BSA PBS was then added to each tube, gently vortexed and centrifuged at 400 × g for 15 minutes at 4°C. Tubes were then resuspended in 1 mL of 1% BSA PBS with 1 μL of sytox orange (Thermo Fisher Scientific, S34861) and incubated on ice for 20 minutes. Data was then acquired using the Attune NxT flow cytometer.
Data was analysed using FlowJo with the following gates. The cell population was first gated on FSC-A vs SSC-A, and single cells were then selected by FSC-A vs FSC-H. Viable cells were identified by gating the sytox orange negative population using YL1-A vs SSC-A and within that gate, KO and WT cells were isolated by BL1-A vs VL1-A using a quadrant gate. Antibody staining was then quantified in the RL1-A channel and histograms were merged to demonstrate the staining intensity between the two populations compared to their two respective controls (secondary only or fluorescence minus one). The figure was then assembled using Adobe Illustrator 2024.
Data availability
Acknowledgments
This is a summary of independent research funded by both the NC3Rs and MRC and carried out at the National Institute for Health and Care Research (NIHR) Leicester Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NC3Rs, the MRC, the NIHR or the Department of Health and Social Care.
We gratefully acknowledge the support of Dr. Carl Laflamme and Dr. Riham Ayoubi, whose technical expertise was invaluable to this work.
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Michael Biddle and Harvinder Virk have received funding for a research studentship from Abcam Ltd, as well as in-kind contributions from manufacturers that contribute to the YCharOS Inc. consortium. The remaining authors declare no competing interests. These relationships did not influence the study design, data collection or analysis, decision to publish, or preparation of the manuscript.
This work was supported by a grant from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and MRC (NC3Rs Ref: NC/NAM0019/1, MRC UKRI076) alongside support from the Leicester Institute for Precision Health.
The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
© 2026 Biddle M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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