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Af933

Manufactured by R&D Systems
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The AF933 is a laboratory instrument used for the detection and quantification of specific biomolecules. It functions as an analyzer, providing users with data and measurements related to the target analytes.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (4 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Ab 108 c, by R&D Systems (3 mentions) Ab49900, by Abcam (3 mentions) Ab92323, by Abcam (3 mentions) Trypan blue, by Merck Group (2 mentions) A9202, by Merck Group (2 mentions) Rhm csf, by Thermo Fisher Scientific (2 mentions) Facsaria fusion, by BD (2 mentions) Nitrocellulose membrane, by R&D Systems (2 mentions) Nupage bis tris protein gel, by Thermo Fisher Scientific (2 mentions) Chemidoc xr, by Bio-Rad (2 mentions) Epr3861, by Abcam (2 mentions) Mouse anti spike s2 1a9, by GeneTex (2 mentions) Epr4435, by Abcam (2 mentions) Ab179707, by Novus Biologicals (2 mentions) A11087, by Thermo Fisher Scientific (2 mentions) Alexa 488, by Thermo Fisher Scientific (2 mentions) Anti vinculin, by Proteintech (2 mentions) Pierce protein free t20 tbs blocking buffer, by Thermo Fisher Scientific (2 mentions)

62 protocols using af933

1

SARS-CoV-2 Spike Protein Binding Inhibition

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Human recombinant full-length ACE2-Fc protein (GenScript, Z03484), Anti-ACE2 Ab (R&D, AF933) and RD#4-anti-Spike Ab (house-made monoclonal antibodies) were used. ACE2-Fc protein was diluted to 20 μg/μl in culture medium and then incubated with SARS-CoV-2 virus solution (MOI = 0.01) at a volume of 1:1 at 37 °C for 30 min. The RD#4-anti-Spike Ab was diluted to 320 ng/μl in culture medium and then incubated with SARS-CoV-2 virus solution (MOI = 0.01) at a volume of 1:1 at 37 °C for 30 min. The virus-ACE2 or virus–antibody mixtures were then added to Jurkat cells or Caco2 cells. Cells were collected for further analysis at 24 h post infection. For anti-ACE2 antibody blocking experiments, Jurkat cells or Caco2 cells were pretreated with 3.33 ng/μl anti-ACE2 antibody (R&D Systems, goat, AF933) at 37 °C for 30 min before infection.
Shen X.R., Geng R., Li Q., Chen Y., Li S.F., Wang Q., Min J., Yang Y., Li B., Jiang R.D., Wang X., Zheng X.S., Zhu Y., Jia J.K., Yang X.L., Liu M.Q., Gong Q.C., Zhang Y.L., Guan Z.Q., Li H.L., Zheng Z.H., Shi Z.L., Zhang H.L., Peng K, & Zhou P. (2022). ACE2-independent infection of T lymphocytes by SARS-CoV-2. Signal Transduction and Targeted Therapy, 7, 83.
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2

Spike Binding and Neutralization Assays

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To allow for Spike binding on the cell surface, CD8+ T cells (0.5×106) were incubated for 30 min at 20°C in 50 μl RPMI-HEPES in the absence of BCS with 2.5 μg Spike Wuhan/BA.1/BA.2 or PBS vehicle control (concentration of Spike treatment chosen based on a dose-response analysis; see Figure S2B). Alternatively, CTLs were pre-treated under the same conditions with 1.2×109/ml MiniVs or control liposomes. Spike-W and the Omicron BA.1 variant were also incubated in combination with the respective neutralizing mAbs (J08 and 02M04, respectively) at a 1:3 protein:antibody ratio. Alternatively, CD8+ T cells (0.5×106) were incubated for 30 min at 20°C in 50 μl RPMI-HEPES in the absence of BCS with 2 μg/ml anti-ACE2 antibody (R&D Systems, #AF933) (Hoffmann et al., 2020 (link)), or the ACE2 substrate/ligand Angiotensin II (Merck, #A9525; 1 μM), or the ACE2 product Ang (1-7) (Sigma, #A9202; 1 μM).
For cytotoxicity assays, cells were pre-treated in the same conditions in serum-free AIM V medium (Gibco, #12055-091) (calcein release assays) or RPMI 1640 (flow cytometry-based assays). None of the treatments affected cell viability at the concentrations and times chosen for the analyses, as assessed by Trypan blue (Sigma-Aldrich, T8154) exclusion (see legend to figures 3 and 5).
Onnis A., Andreano E., Cassioli C., Finetti F., Della Bella C., Staufer O., Pantano E., Abbiento V., Marotta G., D’Elios M.M., Rappuoli R, & Baldari C.T. (2022). SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly. The Journal of Experimental Medicine, 220(2), e20220906.
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3

Western Blot Analysis of ACE2, p38, and NF-κB

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Total proteins were extracted using RIPA buffer (Euromedex, Souffelweyersheim, France), and then equal amounts of proteins were reduced, size-separated on 12% stain-free precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and transferred to nitrocellulose membranes by using an iBlot2 apparatus (Thermo Fisher Scientific). The membranes were blocked in 5% milk in TBS-Tween 0.1% and incubated with specific primary antibodies overnight at 4°C; the antibodies were against ACE2 (AF933, R&D Systems, Minneapolis, MN, USA; 1:200), phospho- and total p38 (9211 and 9212, Cell Signaling Technology, Danvers, MA, USA; 1:2,000), phospho- and total NF-κB p65 (3039 and 8242, Cell Signaling Technology; 1:2,000), and β-actin (A2228, Sigma-Aldrich; 1:5,000). The blots were exposed to horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology, 7074; 1:10,000) and anti-goat (A27104, Thermo Fisher Scientific; 1:2,000) secondary antibodies, and bound antibodies were detected using Clarity chemiluminescent substrate (Bio-Rad). Images were recorded using a Fujifilm LAS-3000 bioimaging system (Stamford, CT, USA).
Ruffin M., Bigot J., Calmel C., Mercier J., Givelet M., Oliva J., Pizzorno A., Rosa-Calatrava M., Corvol H., Balloy V., Terrier O, & Guillot L. (2021). Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression. Frontiers in Immunology, 12, 714027.
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4

SARS-CoV-2 Infection of Alveolar Macrophages

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A bronchoalveolar lavage (BAL) was done in a human lung lobe using ice-cold PBS. BAL cells were filtered and red blood cells lysed. BAL cells were plated in 24 well plates at 5 × 105 cells per well in DMEM, 1% PS, 10% FBS containing 50 ng/ml of rhM-CSF (300–25, Peprotech). For selected experiments, BAL cells were treated for 2h before infection with an ACE2 blocking antibody at 10 μg/ml (AF933, R&D systems). SARS-CoV-2 was added to the cells at MOI 0.1 or 1. After 48h of infection, cells were recovered and stained for viability and surface markers (Supplementary Table 4). After fixation and permeabilization, cells were stained for intracellular spike expression. In selected experiments, AMs (live, EpCAM−, CD3−, CD19−, CD45+, HLA-DR+, CD169+) were flow-sorted prior to infection with SARS-CoV-2 using FACSAria Fusion (BD Biosciences).
Magnen M., You R., Rao A.A., Davis R.T., Rodriguez L., Simoneau C.R., Hysenaj L., Hu K.H., Love C., Woodruff P.G., Erle D.J., Hendrickson C.M., Calfee C.S., Matthay M.A., Roose J.P., Sil A., Ott M., Langelier C.R., Krummel M.F, & Looney M.R. (2022). Immediate myeloid depot for SARS-CoV-2 in the human lung. bioRxiv.
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5

Quantifying Lung hACE2 Levels in Transgenic Mice

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Levels of hACE2 in the lungs of transgenic mice were assessed by Western blotting. Lung cell suspensions were resolved on 4–12% NuPAGE Bis‐Tris protein gels (Thermo Fisher Scientific) and then transferred onto a nitrocellulose membrane (Bio‐Rad, France). The nitrocellulose membrane was blocked in 5% non‐fat milk in PBS‐T for 2 h at room temperature and probed overnight with goat anti‐hACE2 primary Ab at 1 mg/ml (AF933, R&D Systems). Following three wash intervals of 10 min with PBS‐T, the membrane was incubated for 1 h at room temperature with HRP‐conjugated anti‐goat secondary Ab and HRP‐conjugated anti‐β‐actin (ab197277, Abcam). The membrane was washed with PBS‐T thrice before visualization with enhanced chemiluminescence via the super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific) on ChemiDoc XRS+ (Bio‐Rad, France). PageRuler Plus prestained protein ladder was used as size reference. Relative quantification of Western blots was performed using ImageJ program. Images from the same blot were taken with the same exposure time and were inverted before measuring the protein band intensity. The ratio of hACE2 to β‐actin was calculated to indicate the relative expression of hACE2 in each sample.
Ku M., Authié P., Bourgine M., Anna F., Noirat A., Moncoq F., Vesin B., Nevo F., Lopez J., Souque P., Blanc C., Fert I., Chardenoux S., Lafosse L., Cussigh D., Hardy D., Nemirov K., Guinet F., Langa Vives F., Majlessi L, & Charneau P. (2021). Brain cross‐protection against SARS‐CoV‐2 variants by a lentiviral vaccine in new transgenic mice. EMBO Molecular Medicine, 13(12), e14459.
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6

Quantification of SARS-CoV-2 Infection in iAT2 Cells

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For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R&D, #AF933, 4-8 μg/2.5x106 cells) followed by donkey anti-goat IgG-AF647 (Invitrogen, #A21447) or were permeabilized with saponin buffer (Biolegend) then stained with SARS-CoV nucleoprotein (N) antibody (Rockland, #200-401-A50, 1:1000), followed by donkey anti-rabbit IgG-AF488 (Jackson ImmunoResearch, #711-545-152). Gating was based on either mock infected stained controls or infected, isotype-stained controls. Flow cytometry staining was quantified using a Stratedigm S1000EXI and analyzed with FlowJo v10.6.2 (FlowJo, Tree Star Inc). FACS plots shown represent single-cells based on forward-scatter/side-scatter gating.
Huang J., Hume A.J., Abo K.M., Werder R.B., Villacorta-Martin C., Alysandratos K.D., Beermann M.L., Simone-Roach C., Lindstrom-Vautrin J., Olejnik J., Suder E.L., Bullitt E., Hinds A., Sharma A., Bosmann M., Wang R., Hawkins F., Burks E.J., Saeed M., Wilson A.A., Mühlberger E, & Kotton D.N. (2020). SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response. Cell Stem Cell, 27(6), 962-973.e7.
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7

Microvessel Immunofluorescence Imaging

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Microvessels were washed with PBS for 5 minutes, fixed with 3.7% paraformaldehyde (MilliporeSigma) for 15 minutes, blocked with 10% goat serum (overnight, 4°C), then incubated with primary antibody (6 hours, 4°C). Primary antibodies used were rabbit anti-CD31 (Thermo Fisher Scientific RB-10333-P1, 1 μg/mL), goat anti-ACE2 (R&D Systems AF-933, 10 μg/mL), and FITC-conjugated anti-C3c complement rabbit polyclonal antibody (DAKO F0201, 1:75 dilution). After washing with 10% goat serum for 2 hours, the microvessels were incubated (30 minutes, RT) with secondary antibodies (both from Life Technologies, diluted 1:200: Alexa Fluor 647 [A31573 for CD31] and Alexa Fluor 488 [A11055 for ACE2]). Nuclei were visualized with DAPI (Thermo Fisher Scientific). Microvessel cross sections were reconstructed from confocal Z-stacks obtained on a swept field confocal microscope system (Prairie Technologies) with illumination provided by an MLC 400 monolithic laser combiner (Keysight Technologies). For ACE2 images, background subtractions were performed in ImageJ with the background subtract plugin and rolling ball radius set at 500 pixels.
Casciola-Rosen L., Thiemann D.R., Andrade F., Trejo-Zambrano M.I., Leonard E.K., Spangler J.B., Skinner N.E., Bailey J., Yegnasubramanian S., Wang R., Vaghasia A.M., Gupta A., Cox A.L., Ray S.C., Linville R.M., Guo Z., Searson P.C., Machamer C.E., Desiderio S., Sauer L.M., Laeyendecker O., Garibaldi B.T., Gao L., Damarla M., Hassoun P.M., Hooper J.E., Mecoli C.A., Christopher-Stine L., Gutierrez-Alamillo L., Yang Q., Hines D., Clarke W.A., Rothman R.E., Pekosz A., Fenstermacher K.Z., Wang Z., Zeger S.L, & Rosen A. (2022). IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function. JCI Insight, 7(9), e158362.
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8

Detecting Shed ACE2 Fragments in Cell Culture

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After centrifugation to remove insoluble debris and dead cells, cell culture medium was concentrated with the Amicon Ultra-0.5 30K Centrifugal Filter Devices (EMD Millipore, Darmstadt, Germany). The concentrate (25 μl) was then prepared in a buffer consisting of 31.3 mM Tris-HCl (pH 6.8), 1% wt/vol SDS, 5% glycerol, and 0.025% wt/vol bromophenol blue, and boiled for 5 min. Lysates from proximal tubular cells were prepared in a buffer consisting of 62.5 mM Tris⋅HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM DTT, and 0.01% wt/vol bromophenol blue, boiled for 5 min, and centrifuged at 10,000g for 5 min at 4°C to remove insoluble debris.
Twenty-five micro liter of concentrated media (20-fold concentrate) was run on 7.5% SDS-polyacrylamide gels, and subjected to immunoblot analysis using commercially available goat anti-human ACE2 antibodies (1:500 dilution) (AF933, R&D Systems Inc., Minneapolis, MN, USA) as we previously described to characterize mouse shed ACE2 fragments by mass spectrometry (Xiao et al., 2014 (link)). Mouse kidney cortex lysates were used as controls (1.5–10 μg protein). Densitometric analysis of the protein bands was performed using Kodak ID image analysis software (Eastman Kodak, Rochester, NY, USA).
Xiao F., Zimpelmann J., Burger D., Kennedy C., Hébert R.L, & Burns K.D. (2016). Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells. Frontiers in Pharmacology, 7, 146.
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9

Capillary Immunoassay of Transfected Proteins

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One day after transfection, HEK293 cells were lysed in 1xRIPA buffer (Cell Signaling Technology) containing 1 mM PMSF, 1× protease inhibitor cocktail (ThermoFisher Scientific) and soluble protein fractions were isolated by 10 min centrifugation at 12,000g at 4 °C. Using the 12–230 kDa separation module on an automated capillary-based immunoassay system Jess (Protein Simple), approximately 3 µg of lysate from transfected or untransfected HEK cells were analyzed per condition. Samples were incubated with primary antibodies for HA (1:50 dilution, C29F4, Cell Signaling Technology), ACE2 (1:35 dilution, PA520045, ThermoFisher Scientific), ACE2 (1:35 dilution, AF933, R&D Systems), or GFP (1:10 dilution, AF4240, R&D Systems) using the default separation parameters and detected with rabbit or goat chemiluminescent detection modules (Protein Simple).
Meyers W.M., Hong R.J., Sin W.C., Kim C.S, & Haas K. (2023). A cell-based assay for rapid assessment of ACE2 catalytic function. Scientific Reports, 13, 14123.
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10

Quantifying ACE2 Surface Expression

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BHK21 cells were transfected with ACE2 glycosylation mutants with Lipofectamine 3000 (Thermo Fisher Scientific). The cells were harvested at 24 hours post transfection. To determine the cell surface expression level of ACE2, the cells were detached with 10 mM ethylenediaminetetraacetic acid in PBS, fixed in 4% paraformaldehyde, followed by immunolabeling with an antibody against ACE2 (AF933, R&D Systems) without cell permeabilization. The flow cytometry was performed using a BD FACSCanto II flow cytometer (BD Biosciences) and data was analyzed using FlowJo X 10.0.7 (BD). The gating strategy was demonstrated in Supplementary Figure 3.
Chu H., Hu B., Huang X., Chai Y., Zhou D., Wang Y., Shuai H., Yang D., Hou Y., Zhang X., Yuen T.T., Cai J.P., Zhang A.J., Zhou J., Yuan S., To K.K., Chan I.H., Sit K.Y., Foo D.C., Wong I.Y., Ng A.T., Cheung T.T., Law S.Y., Au W.K., Brindley M.A., Chen Z., Kok K.H., Chan J.F, & Yuen K.Y. (2021). Host and viral determinants for efficient SARS-CoV-2 infection of the human lung. Nature Communications, 12, 134.
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