Do more with Less: Improving High Parameter Cytometry Through Overnight Staining

Abstract

Recent advances in flow cytometry have allowed high-dimensional characterization of biological phenomena, enabling breakthroughs in a multitude of fields. Despite the appreciation of the unique properties of antigens and fluorophores in high-parameter panel design, staining conditions are often standardized for short surface stains, regardless of antibody affinity or antigen accessibility. Here, we demonstrate how increasing antibody incubation times can lead to substantial improvements in sensitivity, maintaining specificity, and reducing background, while also significantly reducing the costs of high-parameter cytometry panels. Furthermore, overnight staining reduces the influence of interexperimental variability, assisting accurate pooling over experiments over extended time courses. We provide guidance on how to optimize staining conditions for diverse antigens, including how different fixation strategies can affect epitope accessibility. Overnight staining can thus substantially improve the resolution, repeatability, and cost-effectiveness of high-parameter cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Keywords: antibody incubation; high-parameter cytometry; optimization; overnight staining; spectral cytometry titration.

Figure 1

Influence of time and antibody concentration on mouse CXCR5 staining. (A) Representative histograms of CXCR5 staining on viable C57BL/6 mouse splenocytes after the indicated incubation times. (B) Stain index (n = 5, mean ± SD).

Figure 2

Superior discrimination of human Treg by overnight staining. (A) Representative flow staining of CD4⁺ CD3⁺ cells stained for 30 min or 16 hr. (B) Stain indices of CD25 and CD127 stained for 30 min or 16 hr. Data were acquired on a BD LSRFortessa cytometer.

Figure 3

Intracellular and intranuclear staining can be improved with extended incubation times. (A) Representative staining obtained in 30 min versus 16 hr. (B) Stain indices for Foxp3 and IL‐2. Mouse splenocytes were stimulated and stained for intracellular cytokines as described in the Methods section. Data were acquired on a Cytek Aurora Spectral Cytometer.

Figure 4

Improved detection of CCR7 with overnight intracellular staining. (A) Representative histograms of CCR7 staining on viable C57BL/6 mouse splenocytes after the indicated incubation times. (B) Stain index of CCR7 on CD3⁺ T cells (n = 3, mean ± SD).

Figure 5

Increasing incubation time reduces batch effects. (A) Representative staining and MFI of SIGLEC‐8 on CD45⁺ SSC^hi CD16^‐ cells from the same donor over 3 independent experiments. (B) Representative staining and MFI of CD123 on CD45⁺ SSC^lo CD3⁻ CD19⁻ CD14⁻ CD16⁻ cells from the same donor over 3 independent experiments. (C) Human whole blood immunophenotyping data from the same donor over three independent experiments, stained for 30 min or 16 hr. tSNE plots were generated using the parameters CD45, SSC‐A, CD4, CD8, CD127, CD16, CD19, CD3, CD123, CD20, CD25, Fcer1a, CD11c, SIGLEC‐8, CD56, CD14, and HLA‐DR. Data were acquired on a BD LSRFortessa cytometer. FlowSOM clusters are shown in a colored overlay. (D) Cross entropy distances between samples stained for 30 min or 16 hr. (E) Mouse data from four experiments over the course of two years. Data were acquired on a BD FACSymphony A5 cytometer. tSNE plots were generated using the parameters CD4, CD8, Foxp3, CD103, Neuropilin, CD44, CD62L, Ki67, ICOS, PD‐1, CTLA‐4, CD25, KLRG1, CD69, ST2, and Helios on CD3⁺ T cells. FlowSOM clusters are shown in a colored overlay. (F) Cross entropy distances between mouse samples (intra‐batch variation) or batches (inter‐batch variation). Significance was tested by unpaired t‐test.

Figure 6

Overnight incubation increases cost‐effectiveness. (A) Cost (GBP) per antibody per stain for optimal titration for 1‐hr incubation (median cost £0.21) versus overnight incubation time (median cost £0.04). n = 439, Wilcoxon matched‐pairs signed‐rank test. (B) High‐parameter (23‐50 color) panel costs with 1 hr versus overnight incubation. Paired t‐test.

Figure 7

Enhanced detection of low expression markers or dim fluorophores with overnight staining. (A) CD3 – BV570 staining on viable splenocytes at the indicated times and dilutions. (B) T‐bet – BV605 staining on NK cells. (C) NKp46‐PerCP‐Cy5.5 staining. Examples shown are gated on viable non‐autofluorescent splenocytes.

Figure 8

Reduced non‐specific binding with lower concentrations of antibody in overnight staining. (A) Brilliant Violet dye interactions in a 30 min stain versus an overnight stain. (B) Non‐specific binding of PE‐Cy5 tandems to macrophages in 30 min versus overnight surface staining. Histograms shown are gated on viable F4/80⁺ autofluorescent macrophages.

Figure 9

Protocol overview for optimizing staining conditions.

Figure 10

Titration is essential to maximize sensitivity. (A) Titration of CD3‐Spark Blue 550. (B) Intracellular overnight staining for PD‐1 on viable CD4⁺CD3⁺ T cells at the indicated dilutions on cells fixed and permeabilized with the eBioscience Foxp3 Fix/Perm kit.

Figure 11

Choice of fixative affects staining intensity and specificity. (A) Representative histograms showing overnight CD11b‐BV510 staining on mouse splenocytes either with or without fixation. (B) Stain indices for CD11b staining at various dilutions with various fixatives. (C) Representative examples of overnight marker staining on murine splenocytes with various fixatives.

Figure 12

Controls confirm specificity is maintained with overnight staining. (A) IL‐2 staining on WT or IL‐2‐deficient mouse CD4⁺ T cells. (B) pSTAT5 and Foxp3 staining on mouse CD4⁺ T cells with or without IL‐2 stimulation.

Figure 13

Buffer composition and preparation viability affects cell survival during overnight incubation. (A) Leukocyte (CD45⁺) viability prior to overnight incubation (n = 3, mean ± SD) in mouse spleen or small intestinal lamina propria leukocytes. Statistical analysis by unpaired t‐test. (B) Impact of buffer choice on leukocyte viability in overnight incubation. Cellular viability was assessed on single CD45⁺ leukocytes that were negative for fixable viability dye prior to overnight staining. (C) Viability of various immune cell types from mouse spleen after overnight incubation in different buffers. Statistical analysis for B and C by 2‐way ANOVA with Dunnett's multiple comparisons between PBS FCS and all other conditions.