Isolation of human granulocytes

Materials
	Biocoll
	3.8% citrate solution PBS
	PVA 7200 polyvinyl alcohol 2x PBS, pH 7.4
	Bidest H2O
	Phagocyte buffer (PBS, 0.9 mM CaCl2; 0.5 mM MgCl2; 5 mM glucose; 1% h.i FCS)

Preparations

PVA1% PVA 7200 + 0.9% NaCl

• Add 5 g PVA to 500 ml 0.9 % NaCl solution
• Solve with stirring on a heating plate
• Autoclave
• Cool under stirring
• Store on shelf, protected from light

Procedure

1. Human citrate blood is diluted in PBS (1:1) using 50 ml falcon tubes. The diluted blood is carefully layered over a Biocoll cushion (20 ml diluted blood over 20 ml Ficoll), while not disturbing the Ficoll layer.
2. Centrifuge for 40 min at 20° at 500 g without acceleration (Level 1) & without break (Level 1)
3. After centrifugation, the gray-white granulocyte layer (directly on top of the erythrocyte layer) is carefully collected with a 5 ml pipette (take about 2.5 ml) and transferred to a new 50 ml tube.
4. Mix granulocyte-containing fraction 1:2 with PVA/NaCl (1 part PMNs/2 parts PVA/NaCl) in a 50 ml Falcon tube and mix gently by inverting. Incubate for 30-45 min at RT until the erythrocytes have settled and the solution has clarified. Transfer the clear supernatant containing PMNs into a new tube and centrifuge at RT for 15 min; 300 g with break.
5. After centrifugation, the supernatant is aspirated and the cell pellet is quickly resuspended in 1 ml ddH₂O by swirling the tube. Add 4 ml ddH₂O and swirl the suspension for 40 sec to lyse the remaining erythrocytes. Then, quickly add 5 ml of 2x PBS and centrifuge at RT for 5 min at 200 g with break.
6. Aspirate the supernatant and resuspend the pellet in 5 ml phagocytosis buffer (or any other buffer required by your experiment) and count the granulocytes in a hemocytometer. Set the granulocytes to a cell density of 1 x 106/ml. You should expect to isolate at least 107 granulocytes for every 10 ml of blood.