Related Experiment Videos
Reduced ammonium chloride haemolysis time enhances the number of isolated functional rabbit polymorphonuclear
F Kouoh1, H Levert, B Gressier
1Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, Pharmacocinétique et Pharmacie Clinique, Lille, France.
Abstract:
In the present study we compared seven different methods for isolating rabbit polymorphonuclear neutrophils (PMNs) with a view to assessing viability, lymphocyte contamination and isolation yield. The two methods offering the best isolation yield and functional PMNs were retained. Leukocyte-containing plasma fraction was obtained after erythrocyte sedimentation with dextran. First, PMNs were isolated from this fraction, using hypotonic ammonium chloride haemolysis followed by Histopaque density gradient centrifugation (Method-A). Second, PMNs were obtained from leukocyte-containing plasma after centrifugation on two Percoll layers (Method-B). These processes resulted in a high cellular yield: 2.66x10(6)+/-0.22 PMNs per ml of blood (Method-A) and 1.87x10(6)+/-0.37 PMNs per ml of blood (Method-B). In both cases the PMNs isolated were of high purity and viability. In comparison, when using the standard techniques for rabbit - consisting of ammonium chloride haemolysis taking at least four times as long--fewer PMNs were isolated. The PMNs isolated by Method-A and -B were able to generate a high amount of reactive oxygen species (ROS) after stimulation with phorbol 12-myristate 13-acetate (PMA). These methods to separate PMNs are recommended for in vitro studies.
Insights
This study identifies two optimal methods for isolating rabbit polymorphonuclear neutrophils (PMNs), ensuring high yield, purity, and functionality for in vitro research.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Rabbit polymorphonuclear neutrophils (PMNs) are crucial for studying innate immune responses.
- Efficient isolation of viable and functional PMNs is essential for accurate in vitro research.
- Existing methods for rabbit PMN isolation can be time-consuming and yield suboptimal results.
Purpose of the Study:
- To compare seven methods for isolating rabbit PMNs.
- To identify methods that maximize isolation yield, purity, and viability.
- To evaluate the functional capacity of isolated PMNs.
Main Methods:
- Isolation of leukocyte-containing plasma fraction via dextran sedimentation.
- Method A: Hypotonic ammonium chloride lysis followed by Histopaque density gradient centrifugation.
- Method B: Centrifugation on two Percoll layers.
Main Results:
- Method A yielded 2.66x10^6 +/- 0.22 PMNs/ml blood; Method B yielded 1.87x10^6 +/- 0.37 PMNs/ml blood.
- Both methods produced PMNs of high purity and viability.
- Isolated PMNs demonstrated robust reactive oxygen species (ROS) generation upon PMA stimulation.
Conclusions:
- Methods A and B are highly effective for isolating rabbit PMNs.
- These methods offer superior yield and functionality compared to standard techniques.
- Recommended for in vitro studies requiring pure, functional rabbit PMNs.