ViaKrome Fixable Viability Dye Staining Protocol

Materials

  • PBS
  • Fetal Bovine Serum
  • PerFix-nc Reagent 1, 2 and 3 (Part Number B10825)
  • PBS with 0.5% formaldehyde

Cell Lines

  1. Use cell lines at 5 x 106 cells per mL, 100 μL per test (5 x 105 cells per test)
  2. Add 2.5 uL*of ViaKrome Fixable Viability Dye
  3. Vortex
  4. Incubate at 18-25 °C protected from light for 20 minutes
  5. Add 3 mL of PBS 1X
  6. Centrifuge 5 minutes at 300 g
  7. Aspirate the supernatant
  8. Add 500 μL of PBS 1X / formaldehyde 0.5%
  9. Acquire data using a flow cytometer

*For the use with other protocols and/or samples, a titration of the ViaKrome Fixable Viability Dye is recommended to establish the optimal dose.

Typical Cell Line Staining ViaKrome Fixable Viability Dyes

Representative Staining using ViaKrome Fixable Viability Dyes. Bi-parametric representation (Side scatter versus Fluorescence intensity) of the staining of a mixture untreated cells (Live Cells) and heat-treated Jurkat cells (Dead Cells). Acquisition is done with a CytoFLEX LX N-V-B-Y-R-I Series flow cytometer and emission was detected via the indicated bandpass filter. ViaKrome 405 Dye was excited with the Violet laser (405 nm), panel A, ViaKrome 561 Dye excited with the Yellow Green laser (561 nm), panel B, ViaKrome 638 Dye was excited with the Red laser (638 nm), panel C, and ViaKrome 808 was excited with the Infrared laser (808 nm), panel D.

Peripheral Blood Mononuclear Cells (PBMCs)

  1. Add 2.5 μL of ViaKrome Fixable Viability Dye in 50 μL PBMCs at 10 x 106 cells/mL
  2. Vortex
  3. Incubate at 18-25 °C protected from light for 20 minutes
  4. Add 3 mL PBS 1X
  5. Centrifuge 150 g for 5 minutes. Remove the supernatant by aspiration
  6. Add 50 μL Fetal Bovine serum. Vortex until pellet is dissociated.
  7. Proceed with Intracellular Staining

*For the use with other protocols and/or samples, a titration of the ViaKrome Fixable Viability Dye is recommended to establish the optimal dose.

Typical PBMC Staining ViaKrome Fixable Viability Dyes

Representative Staining using ViaKrome Fixable Viability Dyes. Bi-parametric representation (Side scatter versus Fluorescence intensity) of the staining of peripheral blood mononuclear cells (PMBC). Acquisition is done with a CytoFLEX LX N-V-B-Y-R-I Series flow cytometer and emission was detected via the indicated bandpass filter. ViaKrome 405 Dye was excited with the Violet laser (405 nm), panel A, ViaKrome 561 Dye excited with the Yellow Green laser (561 nm), panel B, ViaKrome 638 Dye was excited with the Red laser (638 nm), panel C, and ViaKrome 808 was excited with the Infrared laser (808 nm), panel D.

 

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