FNA biopsies were collected from four mice bearing A20 tumors, a CD20+ B cell lymphoma model. The samples were stained with a mix of fluorescently labeled anti-CD20 PerCP/Cyanine 5.5 antibody (clone SA275A11) and TotalSeq-B0301 to B0304 anti-mouse Hashtag antibodies (a cocktail of DNA-barcoded anti-CD45 (clone 30-F11) and anti-MHC I clone (M1/42) antibodies in order to doubly label cells to increase tagging efficiency) (both Biolegend) according to the manufacturer’s protocol. The four hashtagged samples were pooled, stained with DRAQ7 viability dye (Abcam) and enriched for immune cells by sorting for CD20− cells on a BD FACSAria III cell sorter (BD Biosciences).
Draq7 viability dye
Manufactured by Abcam
DRAQ7 is a far-red fluorescent cell-impermeable dye that can be used to label dead cells. It is excited by commonly available lasers and has an emission maximum at 660 nm.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3 cell sorter, by BD (1 mentions)
Melon gel igg purification kit, by Thermo Fisher Scientific (1 mentions)
Incucyte zoom instrument, by Sartorius (1 mentions)
Zoom software, by Sartorius (1 mentions)
Rabbit complement ma, by Cedarlane (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
2 protocols using draq7 viability dye
1
Multimodal Profiling of B-cell Lymphoma
2
Complement-Dependent Cytotoxicity Assay
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The complement-dependent cytotoxicity assay (CDC) was performed with IgG purified from pre-immunized (day 1) and post-immunized (day 126) sera of cynomolgus monkeys using Zeba™ Spin Desalting Columns (Thermo Scientific) and the Melon Gel IgG Purification Kit (Thermo Scientific). CDC activity was evaluated in Jurkat-GFP cells and MCF-7, SHIN3 and OVCAR cells labeled with 5 μM of CFSE (carboxyfluorescein succinimidyl ester, Life Technologies). Cells (6000/well) were incubated with cynomolgus antibodies at various concentrations and DRAQ7 viability dye (Abcam) at 2 μM, in the presence or absence of 1 % of rabbit complement-MA (Cedarlane) for 2 h in 384-well plates (37 °C). Green fluorescence (live cells) and red (dead cells) fluorescence were measured on an Incucyte™ ZOOM instrument (Essen Bioscience), and the number of cells was calculated using ZOOM software (Essen Bioscience). All measurements were performed in duplicate. The percentage of CDC was determined as follows: % of CDC = ((% of dead cells)complement – (% of dead cells)medium) normalized against the control without antibody.
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