PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF8, RBD, PUUV, empty PE-SA) or 1:200 dilution (spike, endemic HCoV spikes). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19+/antigen-PE+ or viable/CD19+/antigen-APC+ were sorted as probe positive. The PE+ and APC+ gates were drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.
Live dead bv510
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead BV510 is a fluorescent dye that can be used to distinguish between live and dead cells in a sample. It binds to DNA and emits a fluorescent signal when excited by a 488 nm laser, with a peak emission at 510 nm. This product can be used in flow cytometry applications to identify and quantify the proportion of live and dead cells in a population.
Automatically generated - may contain errors
Lab products found in correlation
Cd19 pe cy7, by BioLegend (2 mentions)
Cd27 bv605, by BioLegend (2 mentions)
Easysep pan b cell magnetic enrichment kit, by STEMCELL (2 mentions)
Igm apc, by Southern Biotech (2 mentions)
Cd38 bb515, by BD (2 mentions)
Macsquanttyto cartridge sorting platform, by Miltenyi Biotec (2 mentions)
Cd3 bv510, by BD (2 mentions)
Cytofix cytoperm solution kit, by BD (1 mentions)
Anti cd14 apc h7, by BD (1 mentions)
Anti cd107a fitc, by BD (1 mentions)
Facsverse flow cytometer, by FlowJo (1 mentions)
Anti ifn γ pe cy7, by BD (1 mentions)
Anti cd3 percp cy5, by BD (1 mentions)
Fc block anti mouse cd16 32, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Collagenase a, by Merck Group (1 mentions)
4 protocols using live dead bv510
1
Antigen-Specific B Cell Sorting
2
Enrichment and Sorting of SARS-CoV-2 Antigen-Specific B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF7a, ORF8, RBD) or 1:200 dilution (spike). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19+/antigen-PE+ were sorted as probe positive. The PE+ gate was drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.
3
Immunophenotyping of Granulocytic Myeloid-Derived Suppressor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All antibodies were purchased from Biolegend (San Diego, CA, USA), except for anti‐CD14‐APCH7, anti‐CD3‐PerCPCy5.5, anti‐CD107a‐FITC and anti‐IFN‐γ‐PECy7, which were purchased from BD Biosciences (Franklin Lakes, NJ, USA). To determine the frequencies of gMDSCs, peripheral blood mononuclear cells were first stained with live/dead‐BV510 (Life Technologies, Waltham, MA, USA) and surface antibodies and then incubated for 20 minutes at 4°C in the dark. gMDSCs were counted as CD15+HLA−DR−CD14− cells derived from CD33+CD11b+ myeloid cells. For intracellular staining, cells were permeabilized with the Cytofix/Cytoperm Solution Kit (BD Bioscience) and stained with anti‐arginase I‐FITC (R&D, Minneapolis, MN, USA) or anti‐IFN‐γ‐PECy7; a matched isotype antibody was used as a negative control. After staining, cells were fixed in 1% paraformaldehyde and analysed using a FACS Verse flow cytometer and FlowJo software. For morphologic analysis, flow cytometric‐isolated gMDSCs were stained with a Wright‐Giemsa kit (Solarbio, Beijing, China) following the manufacturer's instructions.
4
Lung Immune Cell Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lungs were collected on day 14 after SAL or BLM installation. Briefly, the lungs obtained from each mouse were digested by collagenase A (1 mg/ml; Sigma-Aldrich) for at least 40 min, with shaking at 37°C. Phosphate-buffered saline containing 10% fetal bovine serum was added to inactivate the enzyme, and the solution was passed through a 40-µm cell strainer. Cells (106 cells/ml) obtained from the lungs or BAL were incubated with LIVE/DEAD BV510 according to the manufacturer’s instruction (Life Technologies, Carlsbad, CA, USA) followed by anti-mouse CD16/32 Fc block (eBioscience, San Diego, CA) for 15 min at 4°C. Different cell populations were identified using the following corresponding antibodies: a) Ly6C-FITC, Ly6G-Alexa647, CD11b-Pecy7, CD11c-PECy5, and SinglecF-PE (monocytes, neutrophils, eosinophils, and alveolar macrophages) or b) CD11b-APC, CD11c-PECy5, SinglecF-FITC, CD86 PECy7, and CD206-PE (alveolar macrophage activation). Samples were acquired by BD LSRFortessa X-20 Cell Analyzer (BD Biosciences, San Rose, CA) and then analyzed by FlowJo software (Ashland, OR, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!