The University of Iowa Carver College of Medicine

Flow Cytometry Facility

CARVER COLLEGE OF MEDICINE

RESEARCH ADMINISTRATION

Flow Cytometry Protocols

Preparing Cell Samples for Flow Cytometry

Cell Sample Preparation for Running Samples on Facility Instrumentation
Cell Sample Preparation for Sorting with the Becton Dickinson FACS DiVa
Cell Sample Preparation for Sorting with the Becton Dickinson FACS Aria
Policy for Sorting Live Human Cells, Infectious Agents, Cells Infected with Replication Incompetent Virus, or Cells from Animals Previously Exposed to Infectious Agent

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
TrypLE

Staining for Viability

Cell Fixation

Disaggregation of Clumped Cells

Immunofluorescence Staining

Immunofluorescence Staining

Calcium Flux

Using Calcium Green and Fura Red with 488 nm Excitation

Apoptosis

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

DNA Content and Cell Cycle

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
Hyptonic 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

Intracellular Staining

Links to Other Flow Cytometry Facility Protocols