Introduction

eFluor® Nanocrystals are available as ready-to-use primary antibody conjugates for flow cytometry applications. Their narrow emission spectrum and compatibility with organic fluorochromes allows effortless integration into multicolor flow cytometry experiments.

eFluor® Nanocrystal Staining of Cell Suspensions of Mouse Lymphoid Tissue

Materials

• eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222)
• Purified anti-mouse CD16/CD32 (cat. 14-0161) (93) for blocking Fc-mediated antibody-binding
• eFluor® Nanocrystals conjugated antibody
• 12x75 mm round-bottom test tubes (Falcon, cat. 2008) or 96-well round-bottom microtiter plates (Falcon, cat. 3910)

Method

1. Harvest tissue and prepare a single cell suspension in 10 ml eFluor® NC Flow Cytometry Staining Buffer.
2. Pass cell suspension through a nylon mesh (Falcon, cat. 2350) to eliminate clumps and debris.
3. Centrifuge cell suspension 4-5 minutes (300-400xg) at 4°C, discard supernatant.
4. Resuspend the sample and perform a cell count and viability analysis.
5. Centrifuge cells as above and resuspend at 2x10⁷ cells/ml in eFluor® NC Flow Cytometry Staining Buffer.
6. Pre-incubate the cells with 0.5-1 μg of purified anti-mouse CD16/CD32 per million cells for 5-10 minutes on ice prior to staining.
7. Add 50 μl (1x10⁶ cells) of cell suspension to each tube or well.
8. Combine 5 μl of eFluor® Nanocrystals conjugated primary antibody and recommended quantity of other conjugated primary antibodies in an appropriate volume of eFluor® NC Flow Cytometry Staining Buffer so that final staining volume is 100 μl.
9. Cover samples if photo-labile dyes are included in your experiment and incubate 30 minutes on ice or in the refrigerator.
10. Wash cells 2X with eFluor® NC Flow Cytometry Staining Buffer.
11. Resuspend stained cells in eFluor® NC Flow Cytometry Staining Buffer and analyze samples on a flow cytometer.

eFluor® Nanocrystal Staining of Human Peripheral Blood

Materials

• eBioscience 1X RBC Lysis Buffer (cat. 00-4333)
• eFluor® NC Flow Cytometry Staining Buffer (cat. 00-3222)
• Affinity Purified Human FcγR-Binding Inhibitor (cat. 14-9161)
• eFluor® Nanocrystals conjugated antibody
• 12x75 mm round-bottom test tubes (Falcon, cat. 2008) or 96-well round-bottom microtiter plates (Falcon, cat. 3910)

Method

1. Lyse red blood cells (RBC) using eBioscience 1X RBC Lysis Buffer.
2. Centrifuge cells for 10 minutes (300-400xg) at room temperature, discard supernatant.
3. Wash cells 2X with eFluor® NC Flow Cytometry Staining Buffer (2 ml/wash).
4. Resuspend the sample and perform a cell count and viability analysis.
5. Centrifuge cells and resuspend at 2x10⁷ cells/ml in eFluor® NC Flow Cytometry Staining Buffer.
6. Pre-incubate the cells with 20μl of Affinity Purified Human FcγR-Binding Inhibitor per million cells for 5-10 minutes on ice prior to staining.
7. Add 50 μl (1x10⁶ cells) of cell suspension to each tube or well.
8. Combine 5 μl of eFluor® Nanocrystals conjugated primary antibody and recommended quantity of other conjugated primary antibodies in an appropriate volume of eFluor® NC Flow Cytometry Staining Buffer so that final staining volume is 100 μl.
9. Cover samples if photo-labile dyes are included in your experiment and incubate 15-20 minutes on ice or in the refrigerator.
10. Wash cells 2X with eFluor® NC Flow Cytometry Staining Buffer.
11. Resuspend stained cells in eFluor® NC Flow Cytometry Staining Buffer and analyze samples on a flow cytometer.