How to Activate Human Neutrophils
The Activation of Human Neutrophils
Activation of neutrophils is not an all or nothing phenomenon and each function has its own threshold for a response. Please choose the proper stimulus and proper concentration of the stimulus for your experiment design.
Neutrophils are a very sensitive cell type and can be easily activated by reagents. HemaCare does not send cells that have been activated.
- Culture Medium:
- RPMI-1640 supplemented with 1-10% autologous serum
- Washing buffer:
- PBS without calcium or magnesium
- 0.5% Human Serum Albumin (HSA)
- 2mM EDTA
- PBS with calcium and magnesium
- Trypan Blue
- Cell culture flask or 24-well, 48-well, 96-well cell culture plate
- Examples of cytokines that can be used for stimulus:Interleukin 8 (IL-8)
- Leukotriene B4 (LTB4)
- Platelet-activating factor (PAF)
- Interleukin 1 (IL-1)
- Tumor necrosis factor alpha (TNF-α)
- Formylated peptide fMLP
- Phorbol myristate acetate (PMA)
- PMA with ionomycin
- Always wear personal protective equipment and use universal precaution when working with human-derived biological materials.
- Thaw cryopreserved neutrophils according to the HemaCare “How to Thaw Cryopreserved Cells” protocol.
- Re-suspend the post-thaw neutrophils at 1-5 × 106 cells/mL.
- Evaluate the cell viability and cell yield of the post-thaw neutrophil by 0.4% trypan blue exclusion method, using a hemocytometer and a microscope. Evaluate cell purity of the post-thaw neutrophil by flow cytometer.
- Plate cells at desired concentration. Add stimulus of choice into neutrophil culture as the experimental group. For example, PMA (5ng/mL) or PMA (5ng/mL) + ionomycin (250ng/mL) as the experimental group and PBS with calcium and magnesium as the experimental control group.
- Put the culture system at 37°C in a humid atmosphere with 5% CO2 for 15 min.
- Please note: incubation time will need to be adjusted according to the chosen stimulus and experiment design.
- Assess the activation (calcium mobilization, migration, oxidative burst, phagocytosis, or degranulation) of neutrophil according to experiment design.
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