Antibody labeling kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Antibody Labeling Kit is a set of reagents designed to simplify the process of labeling antibodies or other proteins. The kit provides the necessary components to conjugate a label, such as a fluorescent dye or enzyme, to the target protein. This enables the labeled protein to be used in various applications, such as immunoassays or imaging techniques.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (4 mentions) Axiovert 200m, by Zeiss (4 mentions) Fc block cd16 cd32, by BD (3 mentions) Cryostat tissue slice instrument, by Leica (2 mentions) Lsrfortessa, by BD (2 mentions) Active caspase 3, by BD (2 mentions) Axiovision 4, by Zeiss (2 mentions) Tissue tek o c t embedding compound, by Electron Microscopy Sciences (2 mentions) E cadherin decma 1, by BioLegend (2 mentions) Vimentin sc 7557, by Santa Cruz Biotechnology (2 mentions) Neu sc 284, by Santa Cruz Biotechnology (2 mentions) Bio spin p30 columns, by Bio-Rad (2 mentions) Cd45 30 f11, by BioLegend (2 mentions) Ki67 sola15, by BD (2 mentions) Phosphorylated plcγ2 k86 1161, by BD (2 mentions) M4g3ln, by BD (2 mentions) Erk1 2, by BD (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Texas red dextran, by Thermo Fisher Scientific (2 mentions) Fitc dextran, by Thermo Fisher Scientific (2 mentions)

42 protocols using antibody labeling kit

Identification and Analysis of Memory Th2 Cells

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Mouse memory Th2 cells were stained with anti-IL1rL1(ST2) (DIH9, BioLegend), and two subpopulations (ST2 low , ST2 high ) were purified by FACS. Memory Th2 cells were stimulated with immobilized anti-TCRb for 4 hours. Anti-Amphiregulin (206220, R&D Systems) was conjugated by ourselves using an Antibody Labeling Kit (Thermo Fisher Scientific) and used for intracellular staining of mouse samples. Anti-IL-5 (TRFK5, BioLegend), Anti-IL-4 (11B11, BioLegend) and Anti-IL-13 (eBio13A, eBioscience) were used for intracellular staining of mouse samples. Anti-Siglec-F (E50-2440, BD Biosciences), Anti-CCR3 (83101, R&D Systems), Anti-CD101 (Moushi101, eBioscience) and Anti-CD62L (MEL-14, BioLegend) were used for cell surface staining of mouse samples.
T cells from PBMC and dissociated cells from the ECRS polyps were stained with anti-CD4 (OKT4, BioLegend), anti-CD45RO (UCHL1, BioLegend), anti-CD161 (HP-3G10, BioLegend), anti-CRTH2 (BM16, BioLegend), anti-Lineage Cocktails (BioLegend) and anti-CD127 (A019D5, BioLegend). Anti-Amphiregulin (31221, R&D Systems) was conjugated by ourselves using an Antibody Labeling Kit (Thermo Fisher Scientific) and used for intracellular staining for human samples. Anti-IL-5 (TRFK5, BioLegend) and anti-IFNg (4S.B3, BioLegend) were used for intracellular staining of human samples.

Morimoto Y., Hirahara K., Kiuchi M., Wada T., Ichikawa T., Kanno T., Okano M., Kokubo K., Onodera A., Sakurai D., Okamoto Y, & Nakayama T. (2018). Amphiregulin-Producing Pathogenic Memory T Helper 2 Cells Instruct Eosinophils to Secrete Osteopontin and Facilitate Airway Fibrosis. Immunity, 49(1).

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CD123 Antibody Binding Competition

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MOLM13 and SEM cells were cultured in RPMI1640 (Invitrogen) with 10% FBS (Lonza). The FACS competition assay was performed to quantify the competitive binding of different CD123 antibodies to CD123 on MOLM13 and SEM cell. 6H6 (Biolegend; Catalog #306002), CD123-DART_IgG (in-house proprietary), and isotype (in-house proprietary) antibodies were labeled with AF647 using the antibody labeling kit (Thermo Fisher; Catalog # A20186). In the FACS competition assay, 2.5 μg of unlabeled antibody was first added to 5 × 10⁵ cells and incubated at 4°C for 30 minutes, followed by the addition of 0.5 μg AF647-labeled antibody and incubation at 4°C for 60 minutes. The cells were then washed 3 times with autoMACS running buffer and resuspended in autoMACS running buffer (Miltenyi) for FACS analysis. The AF647 fluorescence was read out using MACSQuant instrument (Miltenyi). The competition assay was performed for each antibody pairs, and the %inhibition was calculated by normalizing mean fluorescence intensity (MFI) signals to the isotype/6H6 group.

Liu S.Q., Grantham A., Landry C., Granda B., Chopra R., Chakravarthy S., Deutsch S., Vogel M., Russo K., Seiss K., Tschantz W.R., Rejtar T., Ruddy D.A., Hu T., Aardalen K., Wagner J.P., Dranoff G, & D’Alessio J.A. (2020). A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy. Cancer immunology research, 9(1), 34-49.

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Platelet Surface Receptor Expression Profiling

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Platelet surface expression levels of the human GPIb-IX-V complex were
determined using the GPIbα-specific monoclonal antibody, AP1, and levels
of expression of the human GPIIb/IIIa complex were determined using the
complex-specific monoclonal antibody, AP2. Both antibodies were purchased from
the BloodCenter of Wisconsin Hybridoma Core (Milwaukee, WI). AP1 was conjugated
to AlexaFluor-647 and AP2 was conjugated to AlexaFluor-488 using an antibody
labeling kit (Thermo Fisher, Waltham, MA). Mouse IgG1/kappa isotype control
antibodies conjugated to AlexaFluor-647 or Alexafluor-488 were purchased from
Biolegend and used as negative controls.
Flow cytometry was performed using whole blood samples diluted 1:10 in
HEPES/Tyrode’s buffer (20mM HEPES, 137mM NaCl, 2.7mM KCl, 1mM
MgCl₂, 5.6mM Glucose, 1 mg/ml bovine serum albumin, pH 7.4). The
diluted whole blood samples were incubated in the dark for 20 minutes at room
temperature with AP1–647, AP2–488, or isotype control antibodies.
To quench the reactions, samples were diluted 10 fold with HEPES/Tyrode’s
buffer and analyzed immediately using an Accuri C6 flow cytometer (BD
Biosciences, San Jose, CA). Median fluorescence intensity of GPIbα and
GPIIb/IIIa were determined using FlowJo Ver10.0 software (FlowJo LLC, Ashland,
OR).

Zwifelhofer N.M., Bercovitz R.S., Weik L.A., Moroi A., LaRose S., Newman P.J, & Newman D.K. (2019). Hemizygosity for the Gene Encoding Glycoprotein Ib beta (GPIbβ) Is Not Responsible for Macrothrombocytopenia and Bleeding in Patients with 22q11 Deletion Syndrome. Journal of thrombosis and haemostasis : JTH, 17(2), 295-305.

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Antibody Labeling Techniques

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Antibodies were conjugated to Alexa Fluor 647 using Antibody Labeling Kit (ThermoFisher), pHrodo iFL Green using Protein labelling kit (ThermoFisher) or Biotin using EZ-Link Micro Sulfo-NHS-Biotinylation kit (ThermoFisher) according to manufacturer’s instructions.

Panova V., Attig J., Young G.R., Stoye J.P, & Kassiotis G. (2020). Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event. PLoS Pathogens, 16(5), e1008605.

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Flow Cytometry of Neuronal Markers

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Flow cytometry was conducted as described previously [4 (link)]. Antibodies against Tuj1 (Covance) and GluR2/3 (Millipore) were conjugated to Alexa Fluor 647 using an antibody labeling kit (Thermo Fisher Scientific). The cells were fixed as described, permeabilized with 0.1% Triton X-100, and incubated overnight with fluorophore-conjugated anti-Tuji1 or anti-GluR2/3. The cells were sorted using a FACSCalibur flow cytometry system (BD Biosciences, Franklin Lakes, NJ, USA).

Shimomura A., Patel D., Wilson S.M., Koehler K.R., Khanna R, & Hashino E. (2015). Tlx3 Promotes Glutamatergic Neuronal Subtype Specification through Direct Interactions with the Chromatin Modifier CBP. PLoS ONE, 10(8), e0135060.

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Platelet Activation Assay Reagents

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Monoclonal antibodies (mAb) specific for human αIIbβ3 (clone 312.2) or glycoprotein VI (GPVI; clone 11a12) were purchased from the Versiti – Blood Research Institute Hybridoma Core (Milwaukee, WI). MAb 312.2 was conjugated to AlexaFluor-488 or AlexaFluor-660, and mAb 11a12 was conjugated to AlexaFluor 647, using an antibody labeling kit (Thermo Fisher, Waltham, MA). The phycoerythrin (PE)-conjugated mAb specific for human P-selectin (anti-human CD62P) was purchased from BioLegend (San Diego, CA). Mouse IgG1/kappa isotype control antibodies conjugated to PE, AlexaFluor-647, or AlexaFluor-488 were purchased from BioLegend and used as negative controls. The AlexaFluor-660-conjugated mouse IgG1/kappa isotype control antibody was purchased from Thermo Fisher. Thrombin receptor activating peptide (TRAP) and collagen-related peptide (CRP) were purchased from the Versiti – Blood Research Institute Protein Core. The thromboxane A₂ mimetic U46619 was purchased from Millipore Sigma (St Louis, MO). Adenosine diphosphate (ADP) was purchased from Chrono-Log Corp (Havertown, PA).

Zwifelhofer N.M., Bercovitz R.S., Cole R., Yan K., Simpson P.M., Moroi A., Newman P.J., Niebler R.A., Scott J.P., Stuth E.A., Woods R.K., Benson D.W, & Newman D.K. (2019). Platelet Function Changes during Neonatal Cardiopulmonary Bypass Surgery: Mechanistic Basis and Lack of Correlation with Excessive Bleeding. Thrombosis and haemostasis, 120(1), 94-106.

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Immunofluorescence Staining of Lymphoid Tissues

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Mediastinal lymph nodes (mLN), Peyer’s patches (PP), or inguinal lymph nodes (iLN) were snap-frozen in OTC-embedding medium (Sakura Finetek, The Netherlands). Tissue sections (7 μm) were prepared on micro-slides using a Zeiss cryostat (Carl Zeiss AG. Germany). Sections were air dried, fixed in acetone and blocked using 5% horse serum and then stained by immunofluorescence against the antigen of interest at 4 °C (Supplementary Table 1). CTA1-DD was identified by a chicken anti-CTA1-DD antibody (Agrisera, Sweden) in combination with goat anti-chicken-DyLight488 or as directly AF488-labeled CTA1-OVA-DD fusion protein (Antibody labeling kit, Thermo Fisher Scientific, USA).
Confocal microscopy was performed at the Center for cellular Imaging (CCI, University of Gothenburg, Sweden) using the Zeiss LSM 510 META system and LSM software (Carl Zeiss, AG, Germany). All images within an experiment were equivalently manipulated using Zen software (Carl Zeiss AG, Germany.) to adjust brightness and contrast. For quantification of color intensity, images were imported into Fiji,^{80 (link)} transformed into gray-scale, B-cell follicles were defined as region of interest and the mean color intensity within the region was measured.

Schussek S., Bernasconi V., Mattsson J., Wenzel U.A., Strömberg A., Gribonika I., Schön K, & Lycke N.Y. (2020). The CTA1-DD adjuvant strongly potentiates follicular dendritic cell function and germinal center formation, which results in improved neonatal immunization. Mucosal Immunology, 13(3), 545-557.

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Immunofluorescence Analysis of SERCA2a in LMCs

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Immunofluorescence experiments in LMCs were performed as described earlier, with some modification^{21 (link)}, to determine SERCA2a expression and localization on ER. Briefly, the treated LMCs were fixed with 2% paraformaldehyde and permeabilized with cooled methanol for 4 min at 4 °C. Cells were then blocked with 1% BSA with 5% goat serum for 1 h at room temperature. Cells incubated SERCA2a antibody (1:100) in blocking buffer for two hours at room temperature. After three washes, cells were incubated with Alexa Fluor 594 (goat anti-rabbit, 1:200) for 1 h at room temperature in the dark. Since SEC61, specific to ER protein antibody is also from the rabbit, we used commercially pre-labeled SEC61 antibody (ab205794, Abcam, Cambridge, MA, USA), generated with an antibody labeling kit (A20181, Thermo Fisher, Waltham, MA, USA) and applied after SERCA2a antibody. Cell were counterstained for the nucleus and mounted using Prolong Gold antifade mounting medium with DAPI. Images were taken with Olympus BX41 fluorescence microscope using UPlanFLN X40 objective (NA = 1.3) (Olympus America, Melville, NY, USA) and SERCA2a fluorescence intensity was measured with Image J.

Lee Y., Chakraborty S, & Muthuchamy M. (2020). Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model. Scientific Reports, 10, 12320.

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Quantifying Surface MHC Class I Presentation

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For steady state Class I, cells infected with shRNA lentivirus were stained with fluorochrome-conjugated Abs including anti-HLA-A,B,C (W6/32, prepared in-house), anti-H-2K^b (HB176, prepared in-house), anti-β2m (BBM.1, prepared in-house), and anti-HLA-A2 (MA2.1, prepared in-house). For SIINFEKL presentation, cells infected with shRNA lentivirus were resuspended in FBS-free acidified RPMI 1640 medium, infected with rIAV at MOI = 10 at 37°C, resuspended in cultu re medium, harvested at indicated time points, and stained with fluorochrome-conjugated Abs including anti-NA (NA2–1C1, prepared in-house), anti-M2 (M2–1C6, prepared in-house) and anti-K^b-SIINFEKL (25D1.16, prepared in-house). ES and Ub were UV inactivated before use to avoid saturation of K^b-SIINFEKL on cell surface. Fluorochrome conjugation using antibody labeling kit (ThermoFisher) was conducted following manufacturer’s instructions. Secondary staining was conducted with Alexa Fluor 647–coupled goat anti-mouse IgG (H+L) (Life Technologies), when necessary. Flow cytometric data were acquired using a BD LSR Fortessa X-20 flow cytometer (BD Biosciences), gated on single cells, and data were analyzed with FlowJo version 9.8.5 software (FlowJo LLC).

Wei J., Kishton R.J., Angel M., Conn C.S., Dalla-Venezia N., Marcel V., Vincent A., Catez F., Ferré S., Ayadi L., Marchand V., Dersh D., Gibbs J.S., Ivanov I.P., Fridlyand N., Couté Y., Diaz J.J., Qian S.B., Staudt L.M., Restifo N.P, & Yewdell J.W. (2019). Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance. Molecular cell, 73(6), 1162-1173.e5.

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Immune Cell Identification and Sorting

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Single cells suspensions were incubated in PBS containing 0.5% FCS and 10 mM EDTA with the indicated antibodies (Supplementary Table 1) for 20 min at 4 °C. For the identification of tetramer-binding CD8⁺ T cells, cells were stained with PE-labeled H-2K^b-TRP2_180–188 tetramer (MBL, Woburn, MA) for 10 min at 37 °C. For discrimination between live and dead cells, single-cell suspensions were stained with AQUA-BV510 or e780 in PBS prior to antibody staining (Molecular Probes) or by adding 7AAD prior to acquisition (Calbiochem). For intracellular or intranuclear staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm (BD Biosciences) or the FoxP3 Transcription Factor (eBiosciences) kits, respectively. The anti-LCMV-NP antibody was conjugated with AF657 or AF594 (Antibody Labeling Kit, ThermoFisher). Cells were acquired with a BD LSRFortessa (BD Biosciences) and data analysis was performed using FACS Diva (BD Bioscience, v8.0.1) and FlowJo (Treestar Inc., v10). Cells were sorted on a Biorad S3 (Biorad) or BD Melody Cell Sorter and data were analyzed using FACSChorus (BD Biosciences, v1.3).

Ring S.S., Cupovic J., Onder L., Lütge M., Perez-Shibayama C., Gil-Cruz C., Scandella E., De Martin A., Mörbe U., Hartmann F., Wenger R., Spiegl M., Besse A., Bonilla W.V., Stemeseder F., Schmidt S., Orlinger K.K., Krebs P., Ludewig B, & Flatz L. (2021). Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment. Nature Communications, 12, 4734.

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