Bright Violet CD86 that Resolves Costimulatory Upregulation Cleanly

August 13, 2026

BlueskyReddit
Bioengineering
Rice University
Postdoctoral Researcher

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Company:

BioLegend

Product Name:

Brilliant Violet 421™ anti-human CD86 Antibody, Clone BU63

Catalog Number:

374212

Clone Number:

Our lab studies immune cell activation and signaling in the context of circulating tumor cells and the metastatic microenvironment. CD86 is one of our core costimulatory readouts for antigen-presenting cell activation in human PBMC-derived populations, and we run it alongside CD69, Ki-67, and phospho-NF-κB p65 to build a picture of how activation state changes under different stimulation conditions.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMCs and PBMC-derived B cells and monocytes, freshly isolated and stained in suspension. Roughly one million cells per stain in a 100 µL volume, both in tubes and in 96-well plate format.

Primary Incubation

: 5 µL per test as supplied (100 tests, 50 µg/mL), in cold FACS buffer (PBS with 2% FBS and 2 mM EDTA), 20–30 minutes at 4 °C protected from light, followed by two washes. Titration confirmed the recommended per-test volume rather than correcting it.

Blocking Agent

BioLegend Human TruStain FcX, 5 µL per test, 10 minutes at 4 °C before adding the antibody cocktail.

Secondary Incubation

N/A — directly conjugated primary.

Tertiary Incubation

N/A.

Detection

Flow cytometry, BV421 excited off the 405 nm violet laser and collected in the standard ~421 nm channel. Analysis in FlowJo with gating on singlets, then live cells by viability dye, then the population of interest

Results Summary

BV421 is a genuinely bright fluorophore and it shows here — CD86-positive cells sit well clear of the negative population, and the upregulation we see on stimulated versus unstimulated samples is a clean, resolvable shift rather than a smear that requires argument to interpret. That brightness matters for CD86 specifically, since resting expression on many populations is low and a dimmer conjugate leaves you gating into background. Clone BU63 gave low nonspecific staining once Fc receptors were blocked, and unblocked controls made obvious how much of the apparent signal on monocytes is Fc-mediated if you skip that step. The reagent titrated predictably, and staining was consistent between tube and plate format. Spillover into the neighboring violet channels behaved as expected and compensated normally with single-stain controls.

Additional Notes

Brilliant Violet polymer dyes require a staining buffer containing the appropriate polymer blocker when combined with other BV or Super Bright dyes in the same panel — without it, dye–dye interactions produce artifacts that look like real double-positive populations. This is the single most common way BV421 panels go wrong and it is not a fault of the antibody. Store at 2–8 °C protected from light and do not freeze. Fc blocking is not optional on myeloid populations with this marker.

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Summary

The Good

Very bright on the violet laser, clean separation even where CD86 expression is modest, works at the stated per-test volume without optimization.

The Bad

: Requires a polymer-blocking staining buffer when combined with other BV or Super Bright reagents, which is an extra consumable and an extra thing to get wrong.

The Bottom Line

A bright, dependable CD86 conjugate that makes costimulatory upregulation easy to call — just buy the staining buffer to go with it.

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