LSK FACS-Sorting

LSK sort 

(3 mice/sample)

Stain panel: 

1) Obtain bone marrow from 3 mice 

2) Crush the bones in 50 mL PBS+5 % FBS (sterile and filtered). Pipette up and down several times the cellular suspension. Filter the cell suspension through a 70μm filter into a 50-mL tube. 

3) Spin down (1200 rpm, 5 min) and remove supernatant. 

4) Resuspend cells in 5 ml of ammonium chloride for 1 min. Add 25 ml PBS+5% FBS, spin down (1200 rpm, 5 min) and remove supernatant. Resuspend cells in 20 mL PBS+5% FBS.

5) Count the cells diluting them 1:100 in Trypan Blue. 

6) Take aliquots for unstained control (500 ml)

7) ENRICHMENT with LS column (Miltenyi): 

- Spin down (1200 rpm, 5 min) and remove supernatant.

- Resuspend cells in 100µl / 100 x 10⁶ cells PBS+5%FBS. Add 2.5µl CD117-beads / 100 x 10⁶ (Shake tube well before).

- Incubate 20 minutes in fridge and shake.

- Wash with 10 mL PBS+5% FBS, spin down (1200 rpm, 5 min) and remove supernatant.

- Resuspend the cells in 3 ml PBS+ 5% FBS/column

- Put a MACS column on the magnet and equilibrate the column with 3 ml PBS+ 5% FBS.

- Add bead-stained cells (3 ml) through the filcon cup. Add 3 x 3 ml PBS+ 5% FBS (let the whole volume (3ml) flow through before adding the next 3 ml).

- Remove the column from the magnet. Add 5 ml PBS+5% FBS, elute the bead-stained cells by pushing out the cells using the plunger into the collection tube (CD117+). Add another 3 ml media and push through once more. (Vf = 8 mL)

- OPTIONAL: Count the cells (dilute 1:25 in Trypan Blue); expect approximately 4-6% recovery.

8) Spin down the cells (1200 rpm 5 min 4ºC) discard the supernatant and resuspend with 50 μL of Ab 1X cocktail the enriched sample.

9) Incubate on ice 15 min. 

10) Wash and spin down.

11) Resuspend the sample in 4 mL of buffer and filter.

12) Lin-Sca-1+c-Kit+ cells (LSK) were sorted using a FACSAria-III (BD Biosciences).

* The samples must be sorted in 700 µl of buffer PBS + 5% FBS for trasplant or RLT with b-mercaptoehtanol (10 μl b-ME per 1 ml Buffer RLT) for RNA extraction.