CytoFlex Flow Cytometer Application Notes

Fixable Viability Dye/SSC dot plot was gated on all events and used to differentiate live from dead cells. Using one of the IR channels for the Live/Dead discrimination preserved traditional channels for marker analysis. Side scatter/forward scatter dot plot was gated on live cells. CD45/Side scatter was gated on live cells. This display was used to define the Lymph gate. CD3/CD16 dot plot was gated on Lymphs. This display was used to define NK Cells (CD3 - ,CD16 + ), NKT (CD3 + ,CD16 + ), Non - NKT (CD3 - , CD16 + ), and T cells (CD3 + ,CD16 - ). CD8/CD4 dot plot was gated on T cells. This display was used to define CD4 + CD8 - and CD4 - CD8 + populations. CD8, an abundant marker, was labeled with AF790 in order to prevent potential spillover from this signal into other channels. CD8 histogram for these population indicates approximately 17% of the T cells are CD8 positive. CD4/CD25 dot plot was gated on CD4 + CD8 - cells. This display was used to define

Table 1. Antibodies Used in Multicolor Staining.

Catalog #/ Part Number

Reagent

Vendor

CD45-Krome Orange

A96416 A66329 IM1238U IM2073U IM0448U

Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter BD Bioscience Beckman Coulter BD Bioscience Beckman Coulter

CD3-APC-Alexa Fluor 750

CD16-PE CD56-PE CD4-FITC CD8-A790* CD25-PC7

IM0102 A52882 A74781 B14807 563779 A66328 563945

HLA-DR-Pacific Blue

CD45RA-APC CD20-BV650 CD19-PC5.5 CD5-BV605

CD14-APC-Alexa Fluor 700 A99020

*Prepared by conjugating anti-CD8 to AF790 using AF 790 Antibody Labeling kit (Molecular Probes)

CD25 low , CD25 mid , and CD25 high populations. CD25 expression level correlates with different Treg functions. A pseudocolor plot was used to draw the gates and the resulting colored populations are shown as well. HLA - DR/CD45RA dot plot was gated on all four defined populations to assess these populations for maturity. CD20/CD19 dot plot was gated on Non NKT cells. This display was used to define the B cell gate. CD19/CD5 dot plot was gated on B cells. Finally, CD14/Side scatter dot plot was used to define the monocyte gate (MONOS). Monocyte phagocytosis/adhesion Assay Utilizing Infrared fluorescing Beads In the immune system, phagocytosis is the primary process by which cells identify, isolate, and destroy invaders by engulfing and digesting them. Several diseases have been linked to defective phagocytic ability. Peripheral blood monocytes are an excellent model system to assess phagocytic function of innate immune cells. This assay demonstrates monitoring phagocytosis in monocytes, identified by the surface marker CD14, utilizing a Jade Green particle, which fluoresces in the near-IR channel (Figure 9).

Figure 9. Monocyte Adhesion and Phagocytosis Assay. Samples were prepared in triplicate for unstained, CD14-FITC (Beckman Coulter Part Number B36630) only, and CD14-FITC and SPHERO™ Fluorescent 2.81 µm Jade Green particles (Spherotech Catalog Number FP-3078- 2). Whole blood was stained with CD14-FITC for 15 minutes followed by 15 minutes lysis with 1 mL VersaLyse Lysing Solution Ready-for-use (Beckman Coulter Part Number IM3648). The lysed samples were then washed once with 3 mL of 1x PBS and centrifuged for 5 minutes at 400 x g. The supernatant was discarded. The pellet was resuspended in 1 mL of 1 x PBS. After resuspension 500 µL (5 x10 6 ) of Jade Green particles was added. All samples were incubated in 37˚ C water bath for 60 minutes and then centrifuged for 5 minutes at 400 x g. The supernatant was discarded. The pellet was resuspended in 1 mL of cold 0.02% EDTA in PBS. Samples were analyzed using the CytoFLEX S Blue-Red-Violet-Infrared Flow cytometer. The collected data were analyzed using CytExpert software v 1.2. Monocyte phagocytosis/adhesion function is assessed by the number of CD14 positive events that are also positive in the PF840 channel (right; orange gated).

Every Event Matters | 16