Reduced ammonium chloride haemolysis time enhances the number of isolated functional rabbit polymorphonuclear neutrophils

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.

Cell Culture on MEMS Platforms: A Review

“…For optical methods, fluorescent sorting in microfluidic devices have been demonstrated, and a microfabricated fluorescence-activated cell sorter (mFACS) has been developed [170], which provides higher sensitivity and no cross-contamination at a lower cost. Other microfluidic cell sorting technologies developed include devices for high content cell analysis and sorting, impedance spectroscopy for cell sorting, magnetic cell sorting (MACS) [171], and selective cell lysis to biochemically separate the blood cell types [172,173]. …”

Blood-on-a-Chip

“…In regular separation procedures, bulk volumes of blood are mixed with bulk volumes of the lysing agent for periods of time long enough to account for diffusion and RBC lysis. At the same time, WBCs are also exposed and may be affected by the lysing solution; shortening the exposure time is of interest for minimizing the chance for WBC alteration (54). Although calculations and experiments show that lysing agent diffusion is usually the limiting factor for speeding up the reaction, it is evident that shortening the diffusion distance would provide significant advantages.…”

Nocardia brasiliensis Induces Formation of Foamy Macrophages and Dendritic Cells In Vitro and In Vivo

“…Bone marrow cells of femurs and tibiae of donor BALB/c mice were collected, treated with ammonium chloride [23] and washed in complete RPMI (RPMI-1640/25 mM HEPES/24 mM bicarbonate supplemented with 50–100 U penicillin/mL, 50–100 µg streptomycin/mL, (all Sigma-Aldrich, St.Louis, MO, USA; RPMI) and 10% heat-inactivated fetal bovine serum (Mediatech, Manassas, VA, USA) or complete advanced DMEM (advanced DMEM medium supplemented with 25 mM HEPES, 50–100 U penicillin/mL, 50–100 µg streptomycin/mL, (all Sigma-Aldrich, St.Louis, MO, USA) and 5% heat-inactivated fetal bovine serum (Mediatech, Manassas, VA, USA). Cells were plated at 1×10 5 cells/cm 2 in complete RPMI or complete advanced DMEM with their respective differentiation factors: 1) 30% L929-conditioned medium for BMDM [24], and 2) 20 ng/mL recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF) and 20 ng/mL interleukin 4 (IL-4) (Peprotech, Rocky Hill, NJ, USA) for BMDC.…”