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Staining Protocols

ThermoFisher Flow Cytometry Guides

Detaching Adherent Cells from Tissue Plates without Trypsin

  • Citric Saline is less harsh than EDTA and trypsin and may yield cells with better viability
  • Accutase (Innovative Cell Technologies)
  • TrypLE (ThermoFisher)
  • Detachin (Genlantis)

Staining for Viability

Cell Fixation

Disaggregation of Clumped Cells


  • 0.02mg/ml DNase I (type IIS) to help eliminate clumping (Sigma-Aldrich Cat. No. D4513-VL).

    Add 0.02mg/ml DNase I (type IIS) to all cell preparation steps, including wash steps, to eliminate free DNA from broken cells that leads to aggregation. Cations must be availible to the DNase in order to work properly, i.e., avoid using EDTA.

  • Accumax, commercial product for keeping cells dissociated.
  • Pluronic F-68 to help eliminate clumping.

    The following suggestion is from Steven Z. Merlin, Columbia University.

    "For cell anti-aggregation reagents, try Pluronic F-68 non-toxic, non-ionic surfactant. It is commonly used in bio-fermenters and I have found it to keep various concentrated samples of primary cells and cell lines from forming aggregates during very long sorts. Also works very well for hepatocytes, IEL's, CHO, fibroblasts, and many other cell types having a propensity to form aggregates. It also works well for analyzing and sorting mixed phytoplankton strains from sea water."

    " Pluronic is sold by ThermoFisher (catalog number 24040032) as a 10% stock solution in a 100mL bottle. Add to your sample to make a final concentration of 1% vol/vol."

Stimulation Guide for Intracellular Staining of Cytokines/Chemokines (courtesy of Biolegend)

Immunofluorescence Staining

Absolute Cell Counts

The Facility's Cytek Aurora makes direct volumetric measurement during sample acquisition, allowing investigators to determine the total cell count per cell sample. The Becton Dickinson flow cytometers do not calculate absolute cell counts (total number of cells per sample). In order to make that calcuation using Becton Dickinson flow cytometers, the total volume of cell sample fluid passing through the instrument during data acquisition must be determined. Using a known concentration of beads mixed into the cell sample, the cell sample fluid volume passing through the instrument during acquistion can be established by counting the beads acquired during data acqusisition. The cells per unit volume can then be calculated from the volume that passed through the instrument. Several vendors sell beads for this purpose.

(A/B) x (C/D) = number of cells per total volume in the sample tube (cell concentration as cells/uL)

  • A = number of vender beads added to cell sample
  • B = total volume of cell sample
  • C = cell count from acquired data
  • D = bead count from acquired data

Calcium Flux


DNA Content and Cell Cycle

Intracellular Staining

Plasma and Serum Prep


Links to Other Flow Cytometry Facility Protocols