Analysis of biological samples in the most physiologically relevant state with minimal sample preparation and manipulation is a key objective in many cell biology workflows. Normally, whole blood samples require time-consuming enrichment and manipulation in preparation for analysis on conventional hydrodynamic focusing flow cytometers. These manipulations can result in loss of rare cell types and undesirable phenotypic changes . Acoustic focusing cytometry, introduced with the Attune Flow Cytometer, allows high sample collection rates without any loss in data resolution, thus eliminating the need for pre-acquisition enrichment and manipulation and helping to enable detection of rare events in a timely manner. In human whole blood, red blood cells outnumber white blood cells ~1,000-fold. This creates two hurdles in attempting to analyze whole blood samples without manipulation: 1) collection of a sufficient number of white blood cell events for statistically meaningful data, and 2) distinguishing white blood cells from red blood cells given the high probability of coincident red blood cell events. This application note outlines a strategy for a no-wash, no- lyse approach to a functional assay using Invitrogen pHrodo BioParticles Conjugates and the Attune NxT Flow Cytometer to assess phagocyte function in human whole blood.
Phagocytic cells are a key component of the innate immune system, serving as a first line of defense against invading pathogens. Neutrophils are the most abundant white blood cells in humans and are often the first cell types recruited to the site of infection where they phagocytose and kill invading bacteria . The significance of neutrophils as a first line of defense against infection is highlighted by certain diseases that result in a reduction of total neutrophil numbers or function, resulting in susceptibility to bacterial infection. There are additional phagocytic cells in blood, including monocytes and dendritic cells. Monocytes mature into cell types such as macrophages or inflammatory dendritic cells upon receiving various stimuli . Dendritic cells are specialized antigen-presenting cells that bridge innate and adaptive immunity. Dendritic cells acquire bacterial antigens through phagocytosis and present these antigens to T cells of the adaptive immune system, which are critical players in mediating protection against certain pathogens . Phagocytosis can be detected using pHrodo BioParticles Conjugates—pH-sensitive reagents that fluoresce upon their ingestion into acidic phagosomes. Characterizing the functional capacity of these phagocytic cell types in a whole blood no-wash, no-lyse assay can save time and reduce the potential artifacts that are introduced in alternative protocols that require red blood cell lysis and multiple purification and wash steps.
*There are a variety of Vybrant DyeCycle dyes with multiple color options.
Whole blood was incubated with various concentrations of pHrodo BioParticles Conjugate for 15–30 min followed by labeling with Vybrant DyeCycle Ruby Stain. Phagocytic cells that take up pHrodo BioParticles Conjugates into their acidic phagosomes acquire a positive signal; this allows comparison of cells that have undergone phagocytosis with those that have not (Figures 1B, 1C). The no-lyse, no-wash pHrodo BioParticles phagocytosis assays are highly specific, as shown by the lack of signal when phagocytosis is inhibited at 4°C (Figure 2, middle column). Furthermore, neutrophils (a highly phagocytic cell type) are the primary cell type that acquires a positive pHrodo dye signal, as expected (blue dots in Figure 2C and 2F).
Figure 2. A no-wash, no-lyse assay for identifying phagocytes in whole human blood on the Attune NxT Flow Cytometer. Whole blood was incubated with or without 5 µg/mL pHrodo Green S. aureus BioParticles Conjugate for 30 min at 4°C or 37°C and 5% CO2, diluted, and then labeled with Vybrant DyeCycle Ruby Stain for 15 min at 37°C and 5% CO2. There is very little background fluorescence present when whole blood is cultured with 5 µg/mL pHrodo Green S. aureus BioParticles Conjugate at 4°C (B, E) compared to the strong signal seen at 37°C (C, F), demonstrating the specificity and efficacy of this reagent in a whole blood no-lyse, no-wash assay.
A dose response to pHrodo Green E. coli BioParticles Conjugate elucidates the presence of multiple phagocyte subpopulations within the three major leukocyte scatter populations (Figure 3). At concentrations of 5 µg/mL pHrodo Green E. coli BioParticles Conjugate or less, neutrophils and monocytes appear to be the primary phagocytic cell types. As the concentration of pHrodo Green E. coli BioParticles Conjugate increases, greater frequencies of neutrophils and monocytes show a positive pHrodo signal, and a population within the standard lymphocyte gate acquires a positive pHrodo signal; these are likely dendritic cells (Figure 3, far right column).
With a 4-laser Attune NxT Flow Cytometer, this assay could be further multiplexed to identify specific cell types and changes in activation state due to phagocytic activity. Additional functional probes could be included to detect oxidative burst, a critical microbicidal function in phagocytes. The Attune NxT Flow Cytometer coupled with Invitrogen reagents allows analysis of biological samples with no manipulation and minimal preparation.
Figure 3. A pHrodo Green E. coli BioParticles Conjugate dose response reveals multiple phagocytic cell types in a human whole blood no-wash, no-lyse assay. Whole blood was incubated with 1, 5, or 15 µg/mL pHrodo Green E. coli BioParticles Conjugate or left untreated for 30 min at 37°C and 5% CO2, diluted, and then labeled with Vybrant DyeCycle Ruby Stain for 15 min at 37°C and 5% CO2. (A) As the concentration of pHrodo BioParticles Conjugate increases, the frequency of phagocytic cells that are positive for pHrodo dye increases along with a shift in green fluorescence MFI at the higher concentrations of pHrodo BioParticles Conjugate. (B) At 15 µg/mL of the pHrodo BioParticles Conjugate, it is evident that there are subpopulations within the granulocyte, monocyte, and lymphocyte gates that are actively phagocytosing pHrodo BioParticles Conjugate.
For Research Use Only. Not for use in diagnostic procedures.