Flow cytometry analysis

Flow cytometry analysis 

To analyze the infiltrating immune cells in the brains after stroke, the mice were transcardially perfused with phosphate-buffered saline (PBS) extensively to remove blood cells in the circulation. The hemispheres from sham surgery animals and the contralateral and ipsilateral hemispheres from poststroke animals were harvested at 72 hours after stroke. The hemispheres were mechanically homo- genized and the infiltrating immune cells were isolated with discontinued Percoll gradients (GE Healthcare). Moreover, to study the tamoxifen induction efficiency and specificity in adult bitrans- genic CCR2-CreER mice, BM, PB, and spleen were harvested. Cell suspensions were stained with fluorochrome-conjugated antibodies including CD45 (30-F11, BioLegend), CD11b (M1/70, BioLegend), Ly-6G (1A8, BD Biosciences), Ly-6C (HK1.4, BioLegend), I-Ab (AF6-120.1, BioLegend), CCR2 (Clone #475301, R&D system), CD115 (T38-320, BD Biosciences), and LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific). Subsequently, the stained suspensions were collected with flow cytometric analyses (Attune, Applied Biosystems) and analyzed with FlowJo v10. 
 

Flow cytometry analysis details protocol 
PART1: Collecting the cells from tissue

1. Transcardially perfuse the animals with phosphate-buffered saline (PBS) extensively to remove blood cells in the circulation.
2. Collect the hemispheres into 6 wells plate
3. Add 2mL of HBSS (Sigma H6648) in each sample in 6 well
4. Mechanically homogenize the hemispheres and filter with 100uM cell strainer (Falcon 352360)
5. Spin down and resuspend the pallet with 4mL of 30% Percoll (GE Healthcare)
   • 10mL of 30% Percoll = 3mL of 100% Percoll + 1mL of 10X HBSS + 6mL of ddH₂O
6. Carefully place the 4mL of 30% Percoll cell mixs on top of 2mL of 70% Percoll.
   • 10mL of 70% Percoll = 7mL of 100% Percoll + 1mL of 10X HBSS + 2mL of ddH₂O
7. Centrifuge at 500rcf, 25 min, 4℃, with low accelerate and NO brake 
8. Remove the lipid layer on top of 30% Percoll
9. Collect the cell layer between 30% Percoll and 70% Percoll
10. Wash the harvested cells with HBSS to remove Percoll 
11. Resuspend the cells into desired volume and start the surface markers staining procedure

Flow cytometry analysis details protocol 
PART2: Surface markers staining

1. Live/Dead staining: Spin down the cells and resuspend the cells with buffer with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, L34965) for 30min on ice.
2. Add the CountBright™ Absolute counting beads (Thermo Fisher Scientific, C36950) into each sample. 
3. Wash the cells and resuspend the cells with buffer contained Fc blocker (Biolegend 101320) for 10min on ice.
4. Stain the cells with surface markers (Please see the original paper for markers clone) for 30min on ice.
5. Wash the cells with buffer and resuspend with desired volume and collect the samples with flow cytometer.

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