Tips and Tricks

Choosing the correct buffer for flow cytometry?

Buffers, buffers and more buffers. How do you know which buffer is the right one to use with your staining protocol? Easy, it all depends. The first question you must ask is where are the markers of interest located, surface expressed or intracellular?

Generalized protocol comparison for flow cytometry

1) Surface staining only,

Surfacestaining 1

Prepare cells> Stain with Ab in flow stain buffer (FSB)> Wash with FSB>Analyze

2) surface + cytoplasmic (which include secreted proteins), and

surface cytoplasm

Prepare cells> Stain with surface Ab in FSB> Wash with FSB> IC Fix buffer> Wash with Perm> Stain in Perm(cytoplasmic Ab)> Wash with Perm Analyze

3) surface + cytoplasmic (which include secreted proteins) + nuclear.

Surface, cytoplasm and nuclear

Prepare cells> Stain with surface Ab in FSB> Wash with FSB> Foxp3/Transcription Factor Fix/Perm buffer> Wash with Perm> Stain in Perm (cytoplasmic plus nuclear Ab)**> Wash with Perm> Analyze

Intracellular stainingIntracellular proteins are broken down into two categories, either nuclear or cytoplasmic (which include secreted proteins) or sometimes even both depending on the protein. The location of the protein of interest will dictate which type of buffer system and protocol is recommended: Intracellular Fixation and Permeabilization Buffer Set (cat 00-8824) or Foxp3/Transcription Factor Buffer Set (cat 00-5523).Visit the website for more information Staining Intracellular Antigens for Flow Cytometry