A single-cell suspension of spleens was obtained by mechanical dissociation. Cells were suspended in flow cytometry buffer (2% FBS, 0.1% NaN3, and 1 mM EDTA in Hanks’ balanced salt solution) at 1 × 107 cells/ml and incubated with Fc block (anti-mouse CD16/32; clone 93, BioLegend) for 10 min before incubation with specific antibodies listed in table S1, except for FcγRIIB staining. Dead cells were stained out with Fixable Viability Dye (Thermo Fisher Scientific). For detection of NP-specific B cells, biotinylated NP-BSA (Biosearch Technologies) was incubated with allophycocyanin (APC)-conjugated streptavidin (BioLegend) before being added to the cell suspension. For active caspase-3 staining, Cytofix/Cytoperm buffer (BD Biosciences) was used according to the manufacturer’s instructions. For Blimp1 and TNFα staining, Foxp3/Transcription Factor Staining Buffer (Invitrogen) was used according to the manufacturer’s instructions. Cells were analyzed with a BD LSR Fortessa X-20 flow cytometer (BD Biosciences) and FlowJo software v10.7.2 (Tree Star) or sorted with a fluorescence-activated cell sorting Aria (BD Biosciences). Sorting purity of FDCs was confirmed by flow cytometry (>80%). Sorted cells then underwent stimulation or were stored in TRIzol reagent (Life Technologies) at −80°C for mRNA extraction. Some experiments were performed with frozen cells.
Allophycocyanin apc conjugated streptavidin
Manufactured by BioLegend
Sourced in United States
Allophycocyanin (APC)-conjugated streptavidin is a fluorescent labeling reagent used in flow cytometry and other applications. APC is a fluorescent protein derived from cyanobacteria that emits light in the far-red/near-infrared spectrum. Streptavidin is a protein that binds to the small molecule biotin with high affinity. The APC-conjugated streptavidin allows for the detection and analysis of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Fc block anti mouse cd16 32 clone 93, by BioLegend (1 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Fluorescence activated cell sorting aria, by BD (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Proox 110 oxygen controller, by Biospherix (1 mentions)
3 protocols using allophycocyanin apc conjugated streptavidin
1
Isolation and Characterization of Splenic Immune Cells
2
Tracking RBC Turnover in Hyperoxia
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A hyperbaric chamber (ProOx 110 oxygen controller, BioSpherix, USA) was kindly provided by the laboratory of Dr. Claudio Franco at Instituto de Medicina Molecular (Lisboa). Mice were kept there for 5 days in a hyperoxic atmosphere (75% O2). After that period, mice returned to normoxia conditions. At this moment, two initial biotin injections (1mg/200 μl) stained all the RBCs that were already in circulation before the hyperoxia condition (termed “biotin high RBCs” in the text). Seven days later, a third biotin injection (0.6 mg/200 μl) labeled all the newborn RBCs formed during the hyperoxia treatment (referred to as “biotin intermediate RBCs” in the text). Both populations were monitored once a week for 31 days by flow cytometer (BD LSRFortessa). To that end, 1 μl of blood was collected from the tail of each mouse by poking with a 25g needle, and stained with allophycocyanin (APC) conjugated streptavidin (Biolegend), which binds the biotin.
3
Quantification of Antigen Binding in CAR-T Cells
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For the antigen binding detection, CD5KO CAR-T and mock T cells (1 × 106) were harvested and incubated for 1 h at 4°C with CD5-hFc-Bio (0.4 μg/mL; Kactus Biosystems), washed twice, and then incubated with allophycocyanin (APC)-conjugated streptavidin (BioLegend, San Diego, CA, USA) before subjected to flow cytometry analysis.
For the competitive binding assay, CD5KO CAR-T and mock T cells (1 × 106) were co-cultured with a pre-mixed solution of FHVH3-hFc (10 μg/mL) or H65-hFc (10 μg/mL) and CD5-His (0.4 μg/mL; ACROBiosystems) for 1 h at 4°C, respectively. After washing twice, the cells were incubated with APC-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and the cells were analyzed using flow cytometry.
For the competitive binding assay, CD5KO CAR-T and mock T cells (1 × 106) were co-cultured with a pre-mixed solution of FHVH3-hFc (10 μg/mL) or H65-hFc (10 μg/mL) and CD5-His (0.4 μg/mL; ACROBiosystems) for 1 h at 4°C, respectively. After washing twice, the cells were incubated with APC-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and the cells were analyzed using flow cytometry.
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