Comparison and discussion of the application of three methods for neutrophil preparation
10.3969/j.issn.1673-4130.2014.16.002
- VernacularTitle:3种获得中性粒细胞方法的比较及应用的探讨
- Author:
Peiran HU
;
Miaona BAI
;
Menglu LI
;
Huan YANG
;
Zhi YAO
;
Ying GAO
- Publication Type:Journal Article
- Keywords:
neutrophil;
peripheral blood stem cell;
myeloid differentiation;
immunomagnetic beads
- From:
International Journal of Laboratory Medicine
2014;(16):2123-2125
- CountryChina
- Language:Chinese
-
Abstract:
Objective To make a comparison for the neutrophils prepared either by induction of differentiation of myeloid leuke-mia cell line,or by separation and purification of peripheral blood cells,or by induction of myeloid differentiation of peripheral blood stem cells.Methods NB4 cells were induced differentiation by 1μmol/L all-trans retinoic acid (ATRA)to mature granulo-cytes;neutrophils were separated and purified from peripheral blood by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies;hematopoietic stem cells were separated and purified from peripheral blood by Percoll gradient centrifugation followed by negative selection using magnetic bead-labeled antibodies,and were induced to myeloid differentiation by GM-CSF and G-CSF.Morphology and purity of neutrophils prepared by these three methods were studied by means of MGG stai-ning.CD18 protein expression and subcellular distribution were studied by means of immunofluorescence staining.Results Purity of neutrophil was above 40% by induction of differentiation of NB4 cells,and was about 90% if purified from peripheral blood,and was above 70% if induced by myeloid differentiation of peripheral blood stem cells.There was no obvious difference for CD18 ex-pression in neutrophils prepared by these three methods,and staining of CD18 had a dotted pattern distributed in these cells.Con-clusion Peripheral blood neutrophils prepared by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies are with high purity and viability which is suitable for immediate test of neutrophils from fresh blood.Neutrophils pre-pared by myeloid differentiation of hematopoietic stem cell are with high viability and last for days,which can be used in long test for neutrphils.
