ESO wikiadmin: Created page with "{| class="wikitable" !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 =..."

2025-02-15T17:06:05Z

Created page with "{| class="wikitable" !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 =..."

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{| class="wikitable"
!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 ===

# 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.
# Centrifuge for 40 min at 20° at 500 g without acceleration (Level 1) & without break (Level 1)
# 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.
# 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.
# 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.
# 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.

Isolation of human granulocytes - Revision history