Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining

Abstract

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

Keywords: fluorescent cellular barcoding; mass cytometry; mass-tag cellular barcoding; permeabilization; saponin.

Figure 1

Transient exposure to low concentrations of saponin allows consistent fluorescent or mass-tag cellular barcoding. U-937 cells were fixed and then barcoded in the presence of the indicated concentration of saponin. Barcoding was performed with either (A) isotopically purified Pd isotopes or (B) a combination of DyLight 800 and Pacific Orange.

Figure 2

Transient partial permeabilization with saponin does not result in significant intracellular antibody staining with fluorescent or mass-tagged reagents. U-937 cells were fixed and treated with indicated saponin concentration and barcoded (representative plots from one of three experiments are shown). After barcoding and washing with cell staining medium, cells were stained with anti-pRb (S807/811) either before or after alcohol permeabilization with 100% methanol. (A) Barcoding with isotopically purified Pd isotopes, followed by staining with Ho165 conjugated anti-pRb (S807/811). (B) Barcoding with DyLight 800 and Pacific Orange, followed by staining with Alexa 647 conjugated anti-pRb (S807/811).

Figure 2

Transient partial permeabilization with saponin does not result in significant intracellular antibody staining with fluorescent or mass-tagged reagents. U-937 cells were fixed and treated with indicated saponin concentration and barcoded (representative plots from one of three experiments are shown). After barcoding and washing with cell staining medium, cells were stained with anti-pRb (S807/811) either before or after alcohol permeabilization with 100% methanol. (A) Barcoding with isotopically purified Pd isotopes, followed by staining with Ho165 conjugated anti-pRb (S807/811). (B) Barcoding with DyLight 800 and Pacific Orange, followed by staining with Alexa 647 conjugated anti-pRb (S807/811).

Figure 3

Surface staining of human bone marrow is equivalent with or without partial permeabilization and mass-tag barcoding. A single aliquot of freshly fixed and frozen human bone marrow was split into three tubes and then stained with a panel of 27 surface markers after (A) no treatment, (B) washing in 0.02% saponin, or (C) Pd isotope barcoding in 0.02% saponin.

Figure 4

Mass-tag cellular barcoding prior to surface staining allows for characterization of subtle differences in monocyte surface marker expression between donors. Peripheral blood samples from 4 donors were barcoded using Pd isotopes in 0.02% saponin and then stained with a panel of 20 surface markers. Staining for the indicated markers is shown for the gated monocyte populations from each patient.