MDSCs were seeded in complete medium at a density of 1 × 106 cells/well of a 12 well plate. Unless otherwise stated, Gemtuzumab Ozogamicin (GO, Pfizer) was added at a concentration of 1 μg/ml and incubated for 48 h at 37 °C and 5% CO2. Analysis of cell death was monitored via propidium iodide (PI) (Sigma) uptake quantified on the CyTOFLEX flow cytometer. The cytotoxicity of unconjugated gemtuzumab antibody (Absolute Antibody) (2 μg/ml) and gemtuzumab ozogamicin (2 μg/ml) was similarly compared. For drug internalisation assays, GO was covalently labelled to AlexaFluor-647 fluorophore, with the Alexa Fluor Protein Labeling Kit (Life Technologies, Carlsbad, USA) as per the manufacturer's instructions. 1 μg/ml of labelled GO was added to cells and incubated on ice for 30 min to allow binding to the CD33 receptor, then at 37 °C at different time points for internalisation. Membrane-bound non-internalised drug was stripped using stripping buffer (0.2 M Glycine HCl, pH 2.2) and the cells analysed by flow cytometry. The MFIs of AlexaFluor-647 was determined using FlowJo (BD Biosciences, formerly developed by FlowJo LLC).
Alexa fluor protein labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor Protein Labeling Kit is a laboratory product designed for the fluorescent labeling of proteins. The kit provides reagents and instructions for covalently attaching Alexa Fluor dyes to proteins, enabling their detection and visualization in various applications.
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Lab products found in correlation
Gemtuzumab ozogamicin, by Pfizer (1 mentions)
Bsa alexafluor647 conjugate, by Thermo Fisher Scientific (1 mentions)
Hemoglobin, by Merck Group (1 mentions)
Transferrin alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Lsrfortessa, by BD (1 mentions)
Amersham typhoon, by Cytiva (1 mentions)
Pronase, by Merck Group (1 mentions)
Sp toyopearl 650m, by Tosoh (1 mentions)
Catalase, by Merck Group (1 mentions)
Cysteamine, by Merck Group (1 mentions)
G2133 10ku, by Merck Group (1 mentions)
11 protocols using alexa fluor protein labeling kit
1
GO Cytotoxicity and Internalization in MDSCs
2
Endocytosis of Fluorescent Cargo in Trypanosomes
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Drug or DMSO treated trypanosomes (5×105/mL) were resuspended in 100 μL serum-free HMI-9 medium. Trypanosomes were incubated with fluorescent protein cargo for 15 min at 37°C, 5% CO2. Proteins used were: (i) BSA-AlexaFluor647 conjugate (50 μg Invitrogen, Eugene, OR)), (ii) transferrin-AlexaFluor647 conjugate (50 μg, Invitrogen, Eugene, OR)) and (iii) Haptoglobin phenotype 1-1-AlexaFluor647 complexed with hemoglobin (Sigma) 1:1 by weight (5 μg) (Guyett et al., 2016 (link)). Haptoglobin was labeled using AlexaFluor protein labeling kit (Life Technologies, Invitrogen, Eugene, OR). Cells were transferred to an ice-water bath, washed with cold PBS/G at 4°C (3000g for 5 min), resuspended in PBS/G (500 mL) containing propidium iodide (3 μM), and analyzed on a flow cytometer (Beckman Coulter Cyan): FlowJo software (FlowJo, LLC) was used to gate trypanosomes based on size and shape (forward and side scatter features). Fluorescence intensity of endocytosed cargo was measured only in viable cells (negative for propidium iodide uptake). FlowJo software was used to determine the median fluorescence intensity of each cargo in cells (at least 15,000 events). Three independent biological experiments were performed. Statistical significance of differences in fluorescence intensities was analyzed by two-tailed Student’s T-test with unequal variance.
3
Quantifying LCMV NP Expression in Liver Cells
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To measure the expression levels of LCMV NP on liver cells, purified anti-NP antibodies from the monoclonal IgG-secreting hybridoma VL4 (kindly given by R.M. Zinkernagel, Institute of Experimental Immunology, Switzerland) were coupled to Alexa Fluor 647 using the Alexa Fluor Protein Labeling Kit (Life Technologies, Rockville, MD). Mechanically disrupted liver cells were separated on a discontinuous 40%/80% Percoll (GE Healthcare Canada) gradient to separate parenchymal from nonparenchymal cells. Parenchymal cells were stained with fluorochrome labeled anti-CD45, 7-AAD, and Alexa647-anti-NP and were analyzed by flow cytometry on a BD LSRFortessa (BD Biosciences) and using FlowJo (Tree Star).
4
Preparation of Shiga Toxin Hybrid Holotoxins
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Recombinant Stx1a and Stx2a were prepared according to published methods19 (link). A hybrid holotoxin composed of Stx1a A-subunit and Stx2a B-subunit (Stx1A2B), a hybrid holotoxin composed of Stx2a A-subunit and Stx1a B-subunit (Stx2A1B), Stx1a B-subunit, and Stx2a B-subunit were prepared according to published methods59 (link). Briefly, Stx1a or Stx2a was incubated in a dissociation solution (6 M urea, 0.1 M NaCl, 0.1 M propionic acid, pH 4) and each dissociated subunit was separated by gel filtration column chromatography (Sephacryl S-200; GE Healthcare Sciences, USA). The prepared Stx1a A-subunit and Stx2a A-subunit were mixed with heterologous B-subunit and dialyzed against 50 mM Tris-HCl (pH 7.4) to combine each subunit. Each combined hybrid holotoxin was further purified by gel filtration column chromatography (Sephacryl S-200). 125I-labeled Stx1a (125I-Stx1a), 125I-labeled Stx2a (125I-Stx2a), 125I-labeled Stx1A2B (125I-Stx1A2B), and 125I-labeled Stx2A1B (125I-Stx2A1B) were prepared by the iodine monochloride method as previously described19 (link). Alexa Fluor 488-labeled Stx1a B-subunit and Stx2a B-subunit were prepared using the Alexa Fluor Protein Labeling Kit (Molecular Probes, USA).
5
Fluorescent Labeling of MiPM-SP Protein
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The purified MiPM−SP protein was labeled with the commercial fluorescent probe Alexa-488 from the Alexa Fluor protein labeling kit (Molecular Probes) according to the manufacturer's instructions. The protein was dialyzed (cut-off 3 kDa, 14–16 h, 4°C) in PBS buffer. The integrity and the fluorescence of the labeled protein (*MiPM−SP) were confirmed by SDS-Page with Coomassie blue staining and by gel-imaging equipment (Amersham Typhoon scanner imaging system) (ex. 488/em. 520).
6
Purification and Labeling of CRT Protein
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C1q and gC1q were purified from human serum and were prepared as previously described (21 (link)). Recombinant human CRT was expressed as N-ter HAT-tagged protein in Rosetta 2 cells (DE3) and purified by nickel-Sepharose chromatography as detailed in Ref. (25 (link)). CRT purified from human placenta (26 (link)) was provided by Gunnar Houen, Statens Serum Institut, Copenhagen, Denmark. To test endoxine contamination, CRT sample was digested by pronase (Sigma-Aldrich) 2 mg/ml in 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, pH 7.4 for 24 h at 37°C, and then pronase was heat inactivated before incubation with cells. Purified CRT was labeled with Alexa fluor 488 (A488) by using Alexa Fluor Protein labeling Kit (Invitrogen) according to the kit’s instructions.
7
Covalent Layering Verification for PEGylated USPIONs
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To verify different steps of the covalent layering process, tests were conducted at different points of the procedure. The synthesis of PEGylated USPIONs had previously been verified in our laboratory through transmission electron microscopy, dynamic light scattering, zeta potential, elemental analysis, and iron quantification 73 (link). To verify that the USPION surface is functionalized with a mixed layer of PEG2000 and amine-terminated PEG3400, analysis of increases in hydrodynamic radius from a 100% PEG 2000 layer to an 80%/20% PEG2000/PEG3400-NH2 layer were measured on a Brookhaven 90Plus (Brookhaven Instrument, Holtsville, NY) using dynamic light scattering (DLS). Zeta potential was used to confirm the modification steps. To verify the attachment of PEI, USPIONs were released before attachment of the antibody, and zeta potential was measured. To confirm attachment of antibodies, the antibodies were fluorescently labeled, and fluorescence was measured upon release from mica and subsequent wash of the jNPs. Antibodies were fluorescently labeled using the Alexa Fluor® Protein Labeling Kit from Invitrogen.
8
Macrophage Uptake of Labeled CRT
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Purified CRT was labeled with Alexa fluor 568 (A568) by using Alexa Fluor Protein labeling Kit (Invitrogen) according to the manufacturer's instructions. THP-1 macrophages in 12-well plates (0.5 × 106 cells/well) were incubated with 0.5 ml of complete RPMI 1640 containing A568-labeled recombinant CRT at 5 and 10 μg/ml. Uptake of the protein was allowed to proceed for 30 min at 37°C and internalization was stopped by rapid washing with PBS. Cells were then harvested with 0.25% trypsin/EDTA and immediately analyzed by flow cytometry. Results are expressed as MFI ratio (MFI of experiment with CRT-A568/MFI of experiment without CRT).
9
Isolation and Characterization of Botulinum Toxins
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Toxins and non-toxic components were purified from the culture fluid of C. botulinum type A strain 62A (ref. 8 (link)). Briefly, the organisms were cultured using a cellophane-tube procedure, and the culture supernatant was collected and concentrated by 60% ammonium sulfate precipitation. After dialysis, toxin-containing solution was applied to a SP-Toyopearl 650 M (Tosoh) column. The M-PTC rich fraction was collected and dialysed against 0.01 M sodium phosphate buffer (pH 6.0). L-PTC rich fraction was collected and further purified by lactose gel (EY Laboratories) column, and dialysed against 0.01 M sodium phosphate buffer (pH 6.0). The L-PTC fraction is considered to contain its dimer form, LL-PTC8 (link)9 (link). The purity of toxins and NAPs were confirmed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE; Supplementary Fig. 9a ). Uncropped images of SDS–PAGE gels are shown in Supplementary Fig. 10 .
For immunofluorescence staining, toxins were labelled with Alexa Fluor dye using the Alexa Fluor protein labeling kit (Invitrogen). After the reactions, we confirmed the degree of labelling: M-PTC, 7.7 mol dye per mol protein; L-PTC, 18.2 mol dye per mol protein.
For immunofluorescence staining, toxins were labelled with Alexa Fluor dye using the Alexa Fluor protein labeling kit (Invitrogen). After the reactions, we confirmed the degree of labelling: M-PTC, 7.7 mol dye per mol protein; L-PTC, 18.2 mol dye per mol protein.
10
3D STORM Imaging of Alexa Fluor 647-Labeled Proteins
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