Cell cycle of DT40 cells by IF and FACS

Inhibitor treatment and release in mitosis

(10-12 h, + 30 mins the next day)

• total cell concentration should be 1-3 x 10⁷ cells per flask
• add a final concentration of 2 µM of 1NM-PP1 to the cells (should not be more than 6-7 x 10⁵ cells/mL)
• incubate cells for 10-12 hours at 39 °C with 5 % CO2
• to release the cells in mitosis wash twice at RT with prewarmed medium (1300 rpm; 5 min)
• resuspend cells in fresh media (no more than 1 x 10⁶ cells/mL) and incubate at 39 °C with 5 % CO2 (T = 0 min)
• take a sample of different timepoints (7.5 min or 10 min after release)

Idea
to try three samples as it is and one more ctrl (if possible) without permeabilization and blocking: should work as well in this case.

Preparation of coverslips

(approx. 1h + 1h drying)

• wash with EtOH, and clean them with KIMTECH tissue;
• optional: wash with milliQ H₂O
• KOH (1 M; 15 min; r.t.)
• rinse properly with plenty of H2O: 2x destilated from pipes; 1x milliQ H₂O
• PLL coating: for bigger cover slips (200 µL; 5 min; on parafilm in wet chamber)
• wash: 3x PBS (5min+ pipet up and down 3times/each)
• dry (r.t., approx. 1 h)

Harvest cells

(approx. 2h – during drying step of PLL)

• add EdU to cells of interest to final concentration of 10 µM (10 mM stock; 30 min; 37 °C)
  • add directly to media and slowly shake properly
• harvest cells / count cells as usual
  • IF: 5 x 10⁶ cells
  • FACS: 5 × 10⁶ cells (0.5 × 10⁶ - 1 × 10⁶ could be enough)
  • total in one Eppendorf: 1-3 x 10⁷ cells
• fixation: (pre-warmed) PFA/PBS (4% ; 10 min)
• wash cells twice in PBS (1500 rpm; 4 min)
  • (second time leave PBS and cells at 4°C until staining, do not spin down pelet)

DT40 cells
Use TBS instead of PBS

Staining

(approx. 2h)

!!! all centrifuging steps: Centrifuge at 1500 rpm; 4 min (Optional: 500 g; 5 min) Add 1% BSA before centrifugation (more than 1 volume); after permeabilization, to keep cells down!!!

• 1x centrifuge, remove supernatant;
• permeabilization: Triton X-100 (1 mL; 0.5 % Triton X-100; 15 min)
• add PBS (1 mL; w/ BSA)
• 1x centrifuge, remove supernatant;
• optional: add PBS (1mL; w/ BSA), 1x centrifuge

• blocking: 1 % BSA in PBS (500 µL; 15 - 30 min; if click-it reaction w/o N_3-)
  

• Click-reaction (100 µl per sample)
  • Master mix : Reagents for 5 samples (prepare master mix in the order below)
  1. sterile or milliQ H₂O: 398 µL (up to 500 µL)
  2. Tris (1M; pH 8.2, 10x): 50 µL - we used pH 7.92 should be ~8
  3. CuSO4 (500x; 1 M): 1 µL
  4. ascorbate (10x ): 50 µL (frozen aliquots in -20, ascorbate)
  5. Alexa 647/488-Azide (500x): 1 µL
     
• add 100 µl per sample and incubate 30-60 min at r.t.

• optional: wash with PBS after the click reaction

• add 1 µg/ml DAPI (stock 1 mg/ml = 200x) in PBS – 180 µl (100-200 µl) per sample, mix by pipetting

• optional: Hoechst (should work as well)

• add appropriate volume of cells on to coverslips
• measure FACS from the rest: add PBS up to 500 – 1 000 µL

NOTE:
If the size of cell pellet is an issue: after spinning out Alexa, pellet can be resuspended directly in DAPI/PBS and it can be immediately submitted to FACS analysis.