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Sample Preparation Guidelines for cFluor Immunoprofiling Kit


For anyone working with the cFluor Immunoprofiling Kit to prepare and acquire whole blood or peripheral blood mononuclear cells (PBMCs), here is Cytek's recommended sample preparation instructions. If you are acquiring the samples on your Cytek Aurora or Cytek Northern Lights cytometer, here are 3 additional items to make your workflow easier:

  1. Import the cFluor fluorescent tags to the fluorescent tag lists in your SpectroFlo Library section. If you already have existing cFluor tags in your library, delete them or overwrite them with the tags in this list.
  2. Download the SpectroFlo Experiment Template for the kit and import it into SpectroFlo.
  3. Download and read the Acquisition Protocol

Please note that this kit is designed for research use only and is not for use in diagnostic or therapeutic procedures.

 

Materials Needed

 

Protocol for Staining PBMCs

Plan on using 100,000 cells for each reference control (14 fluorescence control and 1 unstained control), and 600,000 cells for each multicolor sample.

  1. Transfer cells to plate or tubes. For ViaDye Red Fixable Viability Dye Reference Control and Multicolor samples, wash and re-suspend cells in PBS before staining for dead cells. For other samples, wash and re-suspend in Stain Buffer:

    For Tubes: Add 3 mL Stain Buffer to the cells. Recover the cells with centrifugation at 400 x g, 5 minutes at room temperature. Decant and blot tubes, then re-suspend cells to 100 uL of Stain Buffer or PBS by vortexing the tubes.

    For Plates: Pellet the cells with centrifugation at 400 x g, 5 minutes at room temperature. Decant and re-suspend the cells in 200 ul of Stain Buffer and pipette to mix. Pellet the cells with centrifugation again. Decant and re-suspend cells in 100 ul of Stain Buffer or PBS.

  2. Viability Staining: stain with ViaDye Red Fixable Viability Dye at 1:1000 dilution in PBS for 15 minutes while protected from light. Then wash in Stain buffer.
  3. For Reference Controls: Stain with 5 μl of each reagent.
  4. For Multicolor Samples: Stain cells first with cFluor BYG667 IgD for 10 minutes, then with cFluor BYG575 CCR7 for another 10 minutes. Then add the remaining antibodies and incubate for an additional 20 minutes at room temperature protected from light.
  5. Wash cells with Stain buffer to remove unbound reagents.
  6. Decant and re-suspend stained cells in Stain Buffer, 200 ul for Reference Controls and 400 ul for Multicolor Samples.
  7. Acquire and analyze the samples.


Protocol for Staining Whole Blood

Plan to use 100 ul of whole blood for each Reference Control and for each Multicolor Sample.

  1. Transfer cells to a deep well plate or tubes. 
  2. For Reference Controls: Stain with 5 ul of each reagent.
  3. For Multicolor Samples: Stain cells first with cFluor BYG667 IgD for 10 minutes, then with cFluor BYG575 CCR7, for another 10 minutes. Then add the remaining antibodies and incubate for an additional 20 minutes.
  4. Add 2 ml of 1X FACS Lysing Solution to the cells. Vortex immediately for 5 seconds and incubate in the dark for 10 minutes.
  5. Pellet the white blood cells with centrifugation at 400 x g, 5 minutes at room temperature.
  6. Decant and wash with 3ml of Stain Buffer.
  7. Recover the cells with centrifugation at 400 x g, 5 minutes at room temperature.
  8. Decant and re-suspend stained cells in Stain Buffer, 300 ul for both Reference Controls and Multicolor Samples.
  9. Acquire and analyze the samples