• Flow Cytometry Protocols
• Immune Cell Guide

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

• Hoechst for Viability Discrimination
• Propidium Iodide for Viability Discrimination
• Fixable Dyes - Dead cells allow fixable viability dyes to cross their membranes where they stain intracellular amines that are more abundant in the cytoplasm than the extracellular amines on the surface of live cells. Cells can be formaldehyde fixed post staining. Cells stained with these products can also be run unfixed.
  • BD
  • BioLegend
  • BioRad
  • Biotium
  • Miltenyi
  • Thermo Fisher
  • Cytek

Protocol and calculations using FlowJo

• 70% EtOH Fixation Recommendations
• Paraformaldehyde Fixation
• GFP and Fixation
• Sorting Fixed Cells for RNA Sequencing
• Fix, Perm, Block Protocol courtesy of Thermofisher
• Reasons to Fix courtesy of Thermofisher

• Cytodelics Whole Blood Cell Stabilizer
  • Publication: See Method Details 
• Cell Culture Freezing Medium
• Intact tissue

• 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 available 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."

• Immunofluorescence Staining with or without Red Blood Cell Lysis (courtesy of BioLegend)
• Cell Surface Staining (courtesy of BioLegend)
• Overnight Staining (for better separation and reduced antibody use)

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 calculation 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 acquisition can be established by counting the beads acquired during data acquisition. The cells per unit volume can then be calculated from the volume that passed through the instrument. Several vendors sell beads for this purpose.

• Thermo Fisher CountBright
• Thermo Fisher AccuCheck
• BioLegend Precision Count Beads
• Beckman Coulter Flow-Count Fluorospheres

(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

• Using Calcium Green and Fura Red with 488 nm Excitation
• Ratiometric Calcium Flux using Indo 1

• MC540 Plus Propidium Iodide works well on some cell types and yields dual parameter histograms that look like annexin-V FITC vs. propidium iodide
• Annexin V (FITC) and Propidium Iodide 

• Acridine Orange
• BrdU plus Prodium Iodide
• DAPI
• Hoechst 33258 / Ethidium Bromide for Tracking the Number of Cell Cycles
• Propidium Iodide
  • DNA Content of Yeast
  • Whole Cell Preparation (recommended for apoptosis)
  • Hypotonic Solutions Which Yield Nuclei (not recommended for apoptosis)
    • Simple Hypotonic Recipe 
      simplest of all recipes, works well on cell types like HL-60
    • Hypotonic Recipe Plus Detergent 
      used the same as hypotonic recipe, it renders better staining for some cells
    • More Complex Hypotonic Recipe 
      may stabilize the DNA better than simpler recipes
• Simultaneous Staining with GFP and Propidium Iodide for DNA Content 
• Simultaneous Staining of DNA and Intracellular Bcl-2 Protein

• Simultaneous Surface and Cytoplasmic Staining (for CD3)
• Intracellular Cytokine Staining #1
• Intracellular Cytokine Staining #2
• Table of Intracellular Antigen Staining Methods 
• Becton Dickinson Buffer Compatibility Resource (shows intracellular targets and effects of different fix and perm systems)
• Surface and Intracellular Cytokine Staining for Flow Cytometry (courtesy of BioLegend)
• Intracellular Staining (courtesy of BioLegend)

• Plasma and Serum Prep (courtesy of Thermo)

• Mario Roederer's Website (fluorochrome conjugation to antibody)