GENERATION OF SINGLE CELL SUSPENSION FOR SCRNASEQ ANALYSIS (James Li Lab)
- Preparation: filter buffer1 and resuspension buffer with 0.22um filter.
- Prepare 10 ml cell Resuspension buffer( RB) as following: DMEM/F12 w/o phenol red (9.51 ml) + 2% FBS(200µl) + 2mM EDTA (40µl of 0.5M) + 25mM Hepes (250µl of 1M)
- Prepare 50ml Buffer 1 = PBS with 1%BSA(1.43ml of 35%) + 2mM EDTA (200µl of 0.5M)
- Prepare Papain (3 tubes): add 400µl of HBSS to 50µl 10x papain, warm up in 37˚C water bath.
- Precool solutions on ice: HBSS, Buffer I ( 3 tubes-- 1.5 ml in a 5ml Eppendorf tube), and Resuspension Buffer.
- Precool Bench top Centrifuges to 4˚C.
- Cell dissociation
- Dissect embryos in ice-cold HBSS; dissect out cerebellum. Rinse four times in HBSS in a 24-well plate.
- Cut cerebellum into small pieces (about 1 mm in dimension) with fine scissors in a drop of HBSS (about 50µl) in a dish on an ice tray under the microscope.
- Add 10µl of DNAseI (1mg/ml) to 450µl prewarmed papain.
- Transfer the tissues to papain + DNAseI solution.
- Incubate at 37˚C for 30 minutes with rotation.
- Using P1000 tip to pipet up and down, gradually reduce the distance from the bottom of the tube.
- Use glass pipet with gradually smaller opening to gently pipet tissues up and down to dissociate cells.
- Put 70-µm cell strainer on top of the 5-ml Eppendorf tube containing 1.5 ml of Buffer 1. Pass entire mixture through the strainer into the tube – gently press the strainer and slowly pipetting the liquid.
- Wash cell strainer twice with 500µl buffer 1 each and add it to the 5ml tube. The total volume is 3ml now.
- After filtration, pellet cells in the 5ml Eppendorf tube at 300 x g for 10 minutes at 4˚C.
- Remove the supernatant; gently flick the tube to dislodge the pellet.
- If red cell was observed in pellet, do ASK lysis to remove blood cell.
- Resuspend the pellet in Resuspension Buffer (RB): add 100 µl RB buffer
- Take out 1 tube of 2x LiveDeath Assay Mix from -20C. Add equal amount of PBS. Then add 10µl of cell suspension to mix with 10 µl of 1x LiveDeath Assay Mix; incubate at RT for 10 minutes.
- Add 10 µl mixture to a hemacytometor chamber and image.
Reagents:
Papain (Worthington Biochemical Corporation, #LK003176): Add 1 ml HBSS with Ca+ and Mg+) to 1 vial of Papain; Aliquot 50 µl/tube and store at -20˚C.
Buffer1 (1%BSA and 2mM EDTA in PBS): Add 0.2 ml of 0.5M EDTA and 0.5g BSA to 50 ml of PBS; filter with 0.22 µm filter.
HBSS (Hanks Balanced salt solution, life technologies Cat# 14025-092 )
LiveDeath Assay Mix (2x): mix 10µl EthD-1 (2mM, Invitrogen ), 5µl Calcein (4mM, Invitrogen ), and 2.5µl Hoechst (16.2 mM, Invitrogen ) to a final volume of PBS; aliquot 25µl/tube and store at -20˚C.
MACS Tissue Storage Solution (Miltenyi Biotec, Catalog #130-100-008)
RPMI 1640 Medium, no phenol red (Thermofhisher, Catalog #11835030)
Resuspension Buffer (Lebovitz L15 medium with 2% FBS, 25mM HEPES, 2mM
EDTA)
Lebovitz L15 medium, no phenol red (Gibco, Catalog #21-083-027)
Disposables:
Cell strainer size 100 µm (Corning, Catalog #431752)
5ml Eppendorf Tubes (Fisher, Catalog #05-412-581)
Wide Bore Tips
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