Comparison of two modified techniques for purifying blood eosinophils.
As there is much heterogeneity in the morphology and function of blood eosinophils, comparison of their properties between groups of subjects requires recovering the majority of these cells. In two currently used techniques to isolate eosinophils, blood granulocytes are processed either on Percoll gradients after an incubation of granulocytes with 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or on a magnetic cell sorter (MACS). In this study, these techniques were modified to increase the efficiency of eosinophil recovery. With the Percoll gradients, using 1.078 g/ml as the top gradient instead of 1.082 g/ml doubled the eosinophil recovery from 43 +/- 5.3% (mean +/- SEM) to 86.9 +/- 2.9%, without decreasing the purity (96.1 +/- 1.4% versus 96.2 +/- 0.9%). With a MACS, the neutrophils in granulocytes obtained on Ficoll-Paque (1.077 g/ml) instead of on Percoll gradient 1.082-1.094 g/ml, were tagged with anti-CD16 antibodies and eliminated by passing them through a magnetic field. When blood eosinophils of the same subjects were isolated using the two techniques, similar recovery and purity levels were obtained: Percoll gradients, 72.7 +/- 6.8% and 92.5 +/- 2.2%; MACS, 80.2 +/- 5.1% and 90.4 +/- 3.8%. Eosinophils isolated through the two techniques were also compared for their production of superoxide anion and leukotriene (LT) C4, with and without pre-incubation with cytokines interleukin-3, interleukin-5 and granulocyte-macrophage colony stimulating factor. The release of these products was similar between the two eosinophil preparations under all conditions tested except for interleukin-3 where eosinophils isolated with a MACS produced more LTC4. These results show that both techniques efficiently recover pure eosinophils. Furthermore, cell incubation with 10(-8) M fMLP did not enhance superoxide anion and LTC4 production nor modify the response to cytokines. The two modified techniques are therefore suitable for comparative studies of eosinophils from different groups of subjects.
Laviolette
M
M
Unité de Recherche, Hôpital Laval, Sainte-Foy, Quebec.
Bossé
M
M
Rocheleau
H
H
Lavigne
S
S
Ferland
C
C
D000328:Adult; D000595:Amino Acid Sequence; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D004804:Eosinophils / Q000166:cytology* / Q000378:metabolism; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D017997:Leukotriene C4 / Q000096:biosynthesis; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D017997:Leukotriene C4; D009240:N-Formylmethionine Leucyl-Phenylalanine