Human PMN and MNC were isolated from peripheral blood of healthy volunteers using Polymorphprep (Progen) or Ficoll Paque Plus (GE Healthcare), respectively, as previously described.33 (link), 34 (link) Human macrophages were generated by adherence of PBMC using monocyte‐attachment medium (PromoCell). After attachment, cells were incubated for 24 hours in serum‐free X‐Vivo medium (Lonza). Subsequently, 50 ng/mL M‐CSF (PeproTech) was added for macrophage generation for at least 7 days. Human NK cells were generated from PBMC using the human NK isolation kit (Miltenyi BioTec) according to the manufacturer’s instructions. Normal human serum was prepared by using clotting activator containing S‐Monovette 9 mL Z tubes (Sarstedt).
Protocol detail
Isolation and culture of human PBMCs
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Polymorphprep
Ficoll Paque Plus
monocyte‐attachment medium
X‐Vivo medium
M‐CSF
human NK isolation kit
Blood
Cells
Ficoll
Healthy volunteers
Human
Isolation
M csf
Macrophage
Monocyte
Nk cells
Progen
Serum
Corresponding Organization : Kiel University
Other organizations : University Medical Center Utrecht, Ludwig-Maximilians-Universität München
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Variable analysis
independent variables
- Isolation method for human PMN and MNC (Polymorphprep or Ficoll Paque Plus)
- Addition of M-CSF (50 ng/mL) for macrophage generation
- Isolation of human PMN, MNC, macrophages, and NK cells
- Incubation time for macrophage generation (at least 7 days)
- Serum-free X-Vivo medium for macrophage generation
- Attachment of PBMC using monocyte-attachment medium for macrophage generation
- Clotting activator containing S-Monovette 9 mL Z tubes for normal human serum preparation
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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