NovaFluor Dyes for Immunophenotyping

Figure 6. Stable stain indices and MFI values for NovaFluor dye conjugates stored in fixatives, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (2 x 10⁶ cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01) and concurrently stained against Invitrogen NovaFluor Yellow 610 anti-human CD8 (10 µL; Cat. No. H003T02Y03), and Invitrogen NovaFluor Blue 660-120S anti-human CD4 (4 µL; Cat. No. H001T03B08) on ice, protected from light, for 30 minutes. Cells were fixed for 30 minutes using 50 µL of eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No.00-4222-26) and data was collected immediately afterward. Data from the same set of cells was collected again at 4-hours, 24-hours, and 5 days following staining. Cells were stored at 4°C, protected from light, between collection periods. All data was collected using a 5-laser Cytek Aurora using Cytek assay settings, at 30 µL/minute flow rate and 50,000 events were collected in the lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. (A) Time course stability of cells stained with NovaFluor dye–conjugated antibodies and stored in fixative. (B) Effect of fixation on stain indices and MFI of NovaFluor dye conjugates.

Figure 8. NovaFluor dye conjugates are photostable upon prolonged exposure to light, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (2 x 10⁶ cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01). Individual wells, in a 96 well plate, were concurrently single stained against anti-human CD4 using 4 µL/sample of NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, NovaFluor Yellow 570, NovaFluor Yellow 590, NovaFluor Yellow 610, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Red 660, NovaFluor Red 685, NovaFluor Red 700, and NovaFluor Red 710. Cells were stained on ice, protected from light, for 30 minutes. Cells were then fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26) and split into 3 equal parts. A set of cells were then protected from light and stored at 4°C, a set of cells were protected from light and stored at room temperature, and a set of cells was stored at room temperature and exposed to a 13W light for 18 hours. Following the 18-hour period, data from the 3 sets of cells was collected. All data was collected using a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings and a 30 µL/minute flow rate. 50,000 events were collected in the lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. Cells stored at 4°C were compared to both cells stored at room temperature protected from light and cells stored at room temperature exposed to light. Comparison was made by calculating the change in log median fluorescent intensity using the cells stored at 4°C as a baseline. A change of <±5% was considered acceptable. (A) Representative plots of NovaFluor conjugates when tested for photostability (B) Test results for change in log MFI was compared for cells stained with NovaFluor conjugates and tested for photostability.

Figure 9. Stable stain and separation indices of NovaFluor dye conjugates when stained and stored in a master mix containing CellBlox buffer, studied with 5-laser Cytek Aurora. An antibody master mix consisting of Invitrogen NovaFluor Yellow 610 anti-CD8 (30 µL; Cat. No. H003T02Y03), Invitrogen  NovaFluor  Blue 660-120S anti-CD4 (12 µL; Cat. No. H001T03B08),Invitrogen CellBlox Blocking Buffer (528 µL; Cat. No. B001T03F01), and eBioscience  Flow Cytometry Staining Buffer (30 µL; Cat. No. 00-4222-26) was used to stain normal human peripheral blood mononuclear cells (1 x 10⁶ cells/sample). Antibody conjugates and CellBlox buffer were used at manufacturer suggested concentrations, and flow cytometry staining buffer was used to achieve a final staining volume of 100 µL. Cells were stained on ice, protected from light for 30 minutes, immediately, 4 hours, 24 hours, 5 days, 14 days, and 30 days following formation of the master mix. The antibody master mix was stored at 4°C, protected from light. All cells were from the same donor. All cells were fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). Data was collected by a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings using a 30 µL/minute flow rate and 50,000 events were collected in they lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. (A) Representative plots of NovaFluor conjugates when tested for performance in CellBlox buffer for up to 30 days (B) Test results for stain indices, separation indices, MFI and change in log MFI for cells stained and stored with a mastermix containing  NovaFluor conjugates and CellBlox buffer for up to 30 days.

Figure 10. Comparable similarity indices of NovaFluor dye conjugates with UltraComp eBeads and UltraComp eBeads Plus in comparison to fresh cells, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (1 x 10⁶ cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01). Individual wells, in a 96 well plate, were concurrently single stained against anti-human CD4 using 2 µL/sample of NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, NovaFluor Yellow 570, NovaFluor Yellow 590, NovaFluor Yellow 610, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Red 660, NovaFluor Red 685, NovaFluor Red 700, and NovaFluor Red 710. Cells were stained on ice, protected from light, for 30 minutes. Cells were then fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). The same fluors were used to stain UltraComp eBeads (Cat. No. 01-2222-42) and UltraComp eBeads Plus (Cat. No. 01-3333-42) at the rate of 1 µL/test for 15 minutes, protected from light, on ice. All data was collected using a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings and a 30 µL/minute flow rate. 50,000 events were collected in the lymphocyte scatter gate for cells and 5,000 events were collected for each set of beads. Briefly, similarity indices, a number between 0 and 1 that measures how closely a fluor’s spectral signature is to another fluor, were generated for each NovaFluor dye for freshly stained and fixed cells and compared to spectral signatures from UltraComp and UltraComp eBeads Plus. If the similarity index was 0, the signatures were considered to have no similarity, and if the similarity index was ≥0.99, then the signatures were considered to be identical. This data demonstrates that UltraComp and UltraComp eBeads Plus can be used to unmix cells stained with NovaFluor dyes. (A) Representative plots of NovaFluor conjugates when tested for performance with human peripheral blood mononuclear cells, UltraComp and UltraComp eBeads Plus (B) Test results for separation indices for cells, UltraComp or UltraComp eBeads Plus stained and stored with a mastermix containing NovaFluor conjugates.