June 2018

Application Highlight

A high-throughput PD-1 receptor-occupancy assay using the BD FACSCelesta™ flow cytometer

The immune checkpoint inhibitor PD-1, expressed on the surface of T cells, delivers co-inhibitory signals upon interaction with its ligands, PD-L1 and PD-L2. PD-1 signaling plays an important role in the control of immune responses and the maintenance of T-cell homeostasis. In cancer, PD-1 signaling may go awry, resulting in a progressive loss of T-cell effector activity and ultimately cancer-cell immune escape and tumor progression.

Thus, PD-1 is an important target for research on new cancer therapeutics such as pembrolizumab and nivolumab, monoclonal antibodies that target PD-1. These clinically approved antibodies block the interaction of PD-1 and its ligands and can restore T-cell immunity against cancer cells.

A new BD data sheet describes the process of developing a receptor-occupancy assay to assess the binding of PD-1 blocking antibodies to target cells. The experiments involved testing a range of concentrations of multiple compounds on two different T-cell subsets from several donors. This was achieved using an automated high-throughput microplate-based sampling platform (Figure 1 shows a sample plate testing a range of dilutions of several compounds). All the experiments were carried out on a BD FACSCelesta™ flow cytometer (with 3 lasers and 12 fluorescence parameters) equipped with the High Throughput Sampler (HTS) option.

App Highlight - Fig 1

Figure 1.Example layout for 96-well microplate sample acquisition on HTS
The color scheme represents serial dilutions of anti-PD-1 antibodies, with a fixed amount of competitive PE conjugate anti-PD-1 antibodies.

App Highlight - Fig 2 - Thumb
Figure 2. PE-EH12.1 competes with pembrolizumab for binding to PD-1 on activated T cells
App Highlight - Fig 2 - Large
Figure 2. PE-EH12.1 competes with pembrolizumab for binding to PD-1 on activated T cells

Peripheral blood mononuclear cells (PBMCs) were stimulated overnight with 10 μg/mL of immobilized BD Pharmingen™ Purified NA/LE Anti-Human CD3. The cells were pre-incubated with BD Pharmingen™ Human BD Fc Block and stained as follows in triplicate. To determine whether clone EH12.1 competes with pembrolizumab for binding to PD-1, the cells were stained with BD Pharmingen™ PE Mouse anti-Human CD279 (PD-1), clone EH12.1 (PE-EH12.1) at a fixed saturating concentration of 1.25 μg/mL and with serial two-fold dilutions (ranging from 0.15 μg/mL to 20 μg/mL) of the purified antibodies BD Pharmingen™ Purified NA/LE Mouse Anti-Human CD279 (PD-1) or pembrolizumab (BioVision). The cells were also co-stained with BD Horizon™ BUV395 Mouse Anti-Human CD3 and BD Horizon™ BV510 Mouse Anti-Human CD4 to allow discrimination of CD8+ (Left) and CD4+ (Right) T-cell subsets. Results: Reference binding curves (orange) consist of cells that were stained with PE-EH12.1 and serial dilutions of NA/LE purified anti-PD1, clone EH12.1. PE-EH12.1 staining alone in the absence of purified antibodies represents the maximum binding of PE-EH12.1 (100%). As negative controls, the cells were incubated with PE-EH12.1 and serial dilutions of purified isotype controls, BD Pharmingen™ Purified NA/LE Mouse IgG1, Κ Isotype control (msIgG1) or human monoclonal IgG4 (huIgG4, BioLegend®), which do not react with PD-1 (yellow and grey curves). The results show that EH12.1 (orange) and pembrolizumab (light blue) compete for binding to PD-1 on both CD8+ and CD4+ T cells with relatively similar affinity.

Our first step (detailed in the data sheet) was to identify and validate BD Pharmingen™ PE Mouse Anti-Human CD279 (PD-1), clone EH12.1 (PE-EH12.1) as a detection reagent in the receptor-occupancy assays. After determining the optimal concentration for PE-EH12.1 binding to the target cells, we designed a competition assay between PE-EH12.1 and the therapeutic antibodies to compare their binding strength or affinity to the same target cells.

In Figure 2, the binding curve (orange) generated from the competition between PE-EH12.1 and purified EH12.1 was used as reference, since these antibodies bind to the same epitope. Notice how the PE-EH12.1/pembrolizumab binding curve (light blue) was almost identical to the reference binding curve for both CD4+ or CD8+ T-cell subsets, suggesting that clone EH12.1 competes with pembrolizumab for binding to PD-1 with similar affinity.

Additional data revealed the distinct binding properties of pembrolizumab and nivolumab to the same target cells. The data sheet also reports how we similarly tested BD Pharmingen™ PE Mouse Anti-Human CD279, clone MIH4 as a detection reagent and obtained quite different results from clone EH12.1.

Download the new PD-1 receptor occupancy data sheet »
Download the BD FACSCelesta brochure »


Tips & Tricks

Use tandem dyes carefully

Tandem dyes such as PE-Cy™5.5 and APC-Cy™7 provide useful options when designing multicolor experiments, as long as you are aware of their characteristics and limitations.

First, remember that compensation requirements for different tandem dye lots—even from the same catalog number—can vary. Each time you receive a new lot, make sure to recheck the compensation.

Second, tandem dyes can be sensitive to environmental conditions. For example, APC-Cy7 and PE-Cy7 tend to degrade with exposure to light, elevated temperature and fixation. Degradation can change their emission profiles and increase spillover, so minimize their exposure to these conditions. While your cells are waiting to be run, keep the tubes on ice or at least cover them with foil.

It also helps to use BD™ Stabilizing Fixative (Cat. No. 338036) for final fixation. Finally, we recommend using BD’s APC-H7 conjugates instead of APC-Cy7. The APC-H7 dyes have similar fluorescence characteristics but are designed to be more stable. Consult the Optical Filter Guide for advice on optimizing detection of these tandem APC dyes with BD Accuri™ flow cytometers.1


Publication Picks

This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.

Elderberries and glioma

Lamy S, Muhire E, Annabi B. Antiproliferative efficacy of elderberries and elderflowers (Sambucus canadensis) on glioma and brain endothelial cells under normoxic and hypoxic conditions. J Funct Foods. 2018;40:164-179. Full Text

Leukocyte isolation methods

Samaï HC, Rioult D, Bado-Nilles A, et al. Procedures for leukocytes isolation from lymphoid tissues and consequences on immune endpoints used to evaluate fish immune status: A case study on roach (Rutilus rutilus). Fish Shellfish Immunol. 2018;74:190-204. Abstract

Anti-proliferative effects of isorhamnetin

Wei J, Su H, Bi Y, Li J, Feng L, Sheng W. Anti-proliferative effect of isorhamnetin on HeLa cells through inducing G2/M cell cycle arrest. Exp Ther Med. 2018;15:3917-3923. PubMed

Boron-doped bioactive glass nanoparticles

Rad RM, Alshemary AZ, Evis Z, Dilek K, Altunbaş K, Tezcaner A. Structural and biological assessment of boron doped bioactive glass nanoparticles for dental tissue applications. Ceram Int. 2018; doi: 10.1016/j.ceramint.2018.02.230. Abstract

1 All reagents and kits are compatible with both the BD Accuri™ C6 Plus and the BD Accuri™ C6 flow cytometer systems. Information about BD reagent kits, BD Accuri™ C6 and BD Accuri™ C6 Plus software templates, and BD CSampler™ and BD CSampler™ Plus automation options is available at

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