Table 7.31.1.

Troubleshooting Guide for Isolation by Negative Selection of Human Eosinophils

Problem	Possible cause	Solution
Mononuclear cell contamination	Poor layering on density solution	Check technique of overlaying onto Ficoll-Paque
	Density separation disrupted by braking	Make sure the centrifuge brake is off during density centrifugation
	Mononuclear layer incompletely aspirated	Check technique of aspirating off the mononuclear layer after Ficoll-Paque centrifugation
Neutrophil (or mononuclear cell) contamination	Not enough antibody added	Use proper amount of antibody
	Antibody cocktail and/or magnetic colloid are expired	Use fresh antibody cocktail and/or magnetic colloid
	The column was overloaded	Load fewer cells or use two columns
	Excessive flow rate through column applied (may result in a brown- tinged pellet due to the presence of magnetic colloid)	Reload the eluted cells onto the column and apply the recommended flow rate
Red blood cell contamination	Sedimentation and antibody cocktail are inadequate to eliminate red blood cells	Use red blood cell lysis protocol
Platelet contamination	Blood drawn with small- bore needle	Use 19-G needle
	Blood drawn with vigorous traction applied to syringe plunger	Draw blood more gently
	Not enough anticoagulant used	Increase anticoagulant
	Ice-cold HBSS was not calcium- and magnesium-free	Prepare HBSS properly
Poor cell viability	Improper solution pH or contaminated solutions or column.	Prepare new solutions; carefully wash the re-used column according to manufacture instructions.