Blood neutrophil collection and processing

Neutrophils were initially isolated from whole blood using polymorphprep™ (Axis-Shield). Whole blood, taken directly into tubes containing 3.8% sodium citrate, an anti-coagulant, at a ratio of 1:9, was layered at 1:1 ratio over polymorphprep™. This was centrifuged at 1800rpm for 40 minutes, with the brake set to zero. Neutrophils were analysed by flow cytometry to establish the percentage of CD66c positive cells. CD66c is a granulocyte marker, which is highly expressed by neutrophils (130). Purity of the enriched neutrophil population was 61.3% (Figure 2-1). The majority of the contaminating cells were red blood cells but of more concern was the potential for monocyte contamination. It has been shown that the presence of monocytes, even in small numbers, can alter the behaviour of neutrophils (131). To ensure that all responses observed were neutrophil specific, magnetic immunoselection was adopted as the neutrophil purification technique (Figure 2-2). This method is described below and yielded 99.8% CD66c positive cells.

Antibody Concentration Host Supplier

Anti- human CD66c PE (monoclonal)

0.125μg per sample Mouse ebioscience IgG1 isotype PE

(monoclonal)

0.125μg per sample Mouse Beckman Coulter

43

Figure 2-1 Neutrophil purity using polymorph preparation method

a) Histogram showing percentage of cells that are CD66c positive measured by flow cytometry. Neutrophils were isolated using the polymorph preparation method. Blue line = neutrophils labelled with a monoclonal antibody to CD66c conjugated to PE. Red line = matched isotype control. In this sample 61.3% of neutrophils expressed CD66c. b) Flow cytometry scatter plot demonstrating the neutrophil population (A) and contaminating cell population (B) on the basis of forward and sidelight scatter properties. c) Neutrophils were spun onto a slide, then stained using a Romanowsky staining protocol, before being imaged using a Leiss microscope. Cells were histologically identified as neutrophils by their multi- lobed nucleus and stain uptake pattern.

0 20 40 60 80 100 SS Lin: SS Lin 0 50 100 150 200 FS Lin: FS Lin 100 101 102 103 FL2 Log: FL2 Log 0 20 40 60 80 100 % of Max 61.3 a. c. b. A" B" CD66c

44 2.2.1 Negative immunoselection methodology

Antibody complexes and dextran-coated magnetic particles were used to isolate the neutrophils by removing unwanted cells from the cell suspension. The antibody complexes link targeted cell populations to the magnetic particles. Labelled cells are pulled to the sides of the tube when the sample is placed in the EasySep™ magnet. Magnetically labelled cells (the positive fraction) remain in the tube while untouched cells (the negative fraction) can be poured into a new tube. For neutrophil isolation, the tetrameric antibody complexes recognize CD2, CD3, CD9, CD19, CD36, CD56, glycophorin A and dextran coated magnetic particles.

2.2.2 Neutrophil ultra-purification by magnetic immunoselection Neutrophils were isolated from the venous blood of healthy adult volunteers. Blood was taken directly into tubes containing 3.8% sodium citrate. Blood was centrifuged at 270g for 20 minutes at 18°c before plasma was aspirated from the top. Plasma was further centrifuged at 1155g for 10 minutes at 18°C to make platelet poor plasma (PPP). To the cell pellet, 6 ml of 6% dextran was added and total volume made up to 50ml with warmed 0.9% sodium chloride. After 25 minutes, the upper layer, which is red cell depleted following dextran sedimentation, was removed and cells pelleted by centrifugation at 185g for 6 minutes. Neutrophils were separated by discontinuous plasma:Percoll gradient centrifugation, pelleted at 420g for 6 minutes and resuspended in RoboSep™ buffer (Stemcell™ Technologies). Ultra-purification of neutrophils was performed using negative immunoselection. Resuspended neutrophils were mixed with EasySep™ Human Neutrophil Enrichment Cocktail (Stemcell™ Technologies) at 50ul/ml cells, mixed well and incubated at room temperature for 10 minutes. EasySep™ Nanoparticles (Stemcell™ Technologies) were then vigorously pipetted before addition of 100ul/ml cells. After mixing well they were incubated at room temperature for 10 minutes. The whole suspension was then made up to a total volume of 2.5ml by the addition of RoboSep™ buffer before being thoroughly mixed and placed into the EasySep™ magnet for 5 minutes. The desired fraction was poured off by inverting the magnet and

45

Figure 2-2 Neutrophil purity using negative immunoselection method

a) Histogram showing percentage of cells that are CD66c positive measured by flow cytometry. Blue line = neutrophils labelled with a monoclonal antibody to CD66c conjugated to PE. Red line = matched isotype control. Following negative immunoselection where antibody complexes link to CD2, CD3, CD9, CD19, CD36, CD56 and glycophorin A and bind to magnetic particles leaving a pure preparation of neutrophils, 99.8% of neutrophils expressed CD66c. b) Flow cytometry scatter plot demonstrating the one cell population on the basis of forward and sidelight scatter properties. c) Neutrophils were spun onto a slide then stained using a Romanowsky staining protocol, before being imaged using a Leiss microscope. Cells were histologically identified as neutrophils by their multi-lobed nucleus and stain uptake pattern.

0 50 100 150 200 FS Lin: FS Lin 0 20 40 60 80 100 SS Lin: SS Lin 100 101 102 103 FL2 Log: FL2 Log 0 20 40 60 80 100 % of Max 99.8 a. c. b. CD66c

46 tube into a new tube, which was then placed into the magnet for a further 5 minutes. This was repeated once, leaving the negatively selected enriched cells in the new tube ready for use.