Protocols
PMN Cell Migration
Materials:
COSTAR transwell permeable support, 5 μM polybcarbonate membrane, 6.5 mm insert, 24 well plate, (Catalog: 3421)
Cell number per well: 1 × 106
Incomplete Low Serum Growth Medium (RPMI 1640 + 0.5 % FBS)
1 mg/ mL Calcein AM
Migration Time: 45 min to 60 min
Procedure:
- Separate PMN cells and resuspend in 1 mL RPMI 1640 + 0.5 % FBS
- Add 8 μL of 1 mg/mL Calcein AM and incubate the cells in the incubator for 35 minutes.
- Collect the cells by spinning down at 300rcf for 3 minutes
- Wash the cells with 10 mL RPMI 1640 + 0.5% FBS and spin down the cells again.
- Resuspend the cells with RPMI 1640 + 0.5 % FBS to have the final cell concentration of 10 × 106 cells/mL
- Add 100 μL of cells into each upper chamber.
- Add 600 μL RPMI 1640 + 0.5 % FBS containing treatment into each lower chamber.
- Put the cells back to incubator and allow them to migrate for 45 to 60 minutes.
- After the incubation, remove the upper chambers and discard
- Collect the cells in the lower chamber by pipeting up and down 10 times in different areas.
- Add 800 μL PBS into each well and pipeting up and down again and combine PBS and cells in one tube.
- Spin the cells down at 1200rcf for 10 minutes, discard supernatant and resuspend the cells with 100 μL PBS.
- Pipet the cells into Greiner 384 well plate and read fluorescence at Ex485/Em538 with 530 nm cutoff.
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