NovaFluor Dyes for Immunophenotyping

Typically, researchers do not combine PerCP-Cy5.5 and PE-Cy5.5 in the same panel due to spillover and compensation errors. Data here demonstrate compensation errors observed when using the tandem dyes PerCP-Cy5.5 and PE-Cy5.5 together in a panel collected on a default Attune cytometer setup (top row). Spillover from PerCp-Cy5.5 into the YL3 channel results in spreading of the PerCP-Cy5.5 negative populations, resulting in populations that do not resolve properly. Incorrect compensation can also lead to downstream analysis errors, as shown in the CD45RA+CD45RO+ population (top row) that does not exist when the CD4 and CD8 populations are separated properly (bottom row) using NovaFluor dyes. NovaFluor dyes that occupy the same primary channels as PerCp-Cy5.5 and PE-Cy5.5 (BL3 and YL3) can clearly resolve cellular populations in our T cells panel, allowing for proper downstream analysis and expanded panel selection.

Figure 2. Use of NovaFluor dyes effectively corrects the compensation errors seen when using conventional fluorophores. Normal human peripheral blood mononuclear cells (1 x 106 cells/well) were stained in 1x PBS using 1 µL of LIVE/DEAD Fixable Violet Dead Cell Stain Kit for 405nm excitation (Cat. No. L34955) for 30 minutes. Cells were washed and then blocked using CellBlox Blocking Buffer (Cat. No. B001T03F01) and Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-71) in eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). Cells were then stained in two separate panels, one using CD4 Monoclonal Antibody (RPA-T4), PerCP-Cyanine5.5 (5 µL, Cat. No. 45-0049-42) and CD8a Monoclonal Antibody (RPA-T8), PE-Cyanine5.5 (5 µL, Cat. No. 35-0088-42), and the other replacing PerCp-Cy5.5 with CD4 Monoclonal Antibody (SK3 (SK-3)) NovaFluor Blue 660-120S (5 µL, Cat. No. H001T03B08) and replacing PE-Cy5.5 with CD8a Monoclonal Antibody (OKT8 (OKT-8)), NovaFluor Yellow 660 (5 µL, Cat. No. H003T03Y04). Cells were stained for 30 minutes at 4°C, washed, and then fixed using 100 µL of eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) for 30 minutes. Cells were then washed, and data was collected on an Attune CytPix Flow Cytometer (4-laser configuration) from 30,000 cells from the lymphocyte gate. Collected data was compensated and later analyzed using FlowJo® software.

Fluorophores designed to avoid cross-laser excitation can be valuable in the expansion of usable detectors on a flow cytometry instrument. NovaFluor dye conjugated antibodies display lower cross-laser spillover in comparison to the conventional PE tandem dye conjugated antibodies.

Figure 3. Spillover reduction using NovaFluor antibodies to replace conventional tandem dyes. UltraComp eBeads Plus Compensation Beads (Cat. No. 01-3333-42) were single stained with either CD4 Monoclonal Antibody (RPA-T4), PE-eFluor 610 (5 µL, Cat. No. 61-0049-42), CD4 Monoclonal Antibody (SK3 (SK-3)) NovaFluor Yellow 610  (5 µL, Cat. No. H001T02Y03), or CD19 Monoclonal Antibody (HIB19), NovaFluor Blue 610-70S (5 µL, Cat. No. H004T03B06). Beads were stained for 30 minutes in eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26), washed, and then analyzed on an Attune CytPix Flow Cytometer (4-laser configuration) (red arrows show spillover). Data shown here are raw fluorescent signal in the BL2 and YL2 channels for each fluorophore. Signal intensity is shown here (x axes) in the YL2 and BL2 channels, demonstrating the strong spillover in BL2 from PE-eFluor 610 dye. This spillover is mitigated with the use of NovaFluor dyes that occupy the same primary channels (BL2, YL2), allowing correct compensation and increased panel size.

Always use CellBlox Blocking Plus Buffer with NovaFluor dyes when labeling cells for best background reduction

See how to use CellBlox Plus Blocking Buffer in the Cell Surface Staining Protocol.

Figure 5. CellBlox Plus Blocking buffer offers improved blocking compared to CellBlox Buffer. Peripheral blood mononuclear cells (PBMCs) were either unblocked (A), blocked with CellBlox Blocking Buffer (B001T06F01) (B), or CellBlox Plus Blocking Buffer (C001T06F01) (C). Cells were then stained with either CD4 monoclonal antibody, NovaFluor Yellow 755 (top) or CD4 monoclonal antibody, NovaFluor Red 710 (bottom). All cells were co-stained with CD19 Monoclonal Antibody (HIB19), eFluor 450 (48-0199-42). Two-dimensional pseudocolor plots of CD4 vs CD19 show that the CellBlox Plus Blocking Buffer reduces non-specific binding compared to staining without CellBlox Plus Blocking Buffer and with CellBlox Blocking Buffer. Data were acquired on a 5-laser Cytek Aurora Flow Cytometer.

Figure 6. CD3 labeling, combined with CellBlox Plus Blocking Buffer, is shown to reduce background in lymphocytes and non-specific labeling of monocytes and macrophages compared with labeling without CellBlox Plus Blocking Buffer. Peripheral blood mononuclear cells (PBMCs) were either left unstained (black), stained with CD3 Monoclonal Antibody (UCHT1), NovaFluor Blue 660 120S conjugate (H002T03B08) with (red), and without (blue) the addition of CellBlox Plus Blocking Buffer (C001T06F01). (A) Forward Scatter vs. Side Scatter pseudocolor plot shows lymphocyte and monocyte gates. Histogram overlay plots show the expression of CD3 within the (B) lymphocyte and (C) monocyte gates. Data were acquired in the B7 channel on a 5-laser Cytek Aurora.