Preparation of Reagents
ATP solution
Adenosin5’-triphosphate disodium salt hydrate (Sigma, A3377)
Water (Sigma, w3500, RNBC5693)
- Dilute 110.57mg of ATP into 1ml of H2O(200nM in H2O)
- Sterilize the ATP solution through a sterilizing filter
- Aliquot in 30μl increments
- Store at -20℃
bFGF solution
bFGF (WAKO, 060-04543, 100μg)
PBS (Gibco, 14170)
- Dilute 100μg of bFGF into 100μl of PBS
- Aliquot in 10μl increments
- Store at -20℃
B27 medium
F12/DMEM 1:1 (Gibco, 11330)
B27 (Gibco, 17504)
LIF (Millipore, ESGRO, mLIF, ESG1107)
- Add 10ml of B27 and 50μl of LIF into 500ml of F12/DMEM medium
- Sterilize the mixture through a sterilizing filter
- Store at 4℃
Materials and Equipment
1 week-old mice (2 to 3 of spleens, or other tissues*)
HBSS (GIBCO, 14170)
Lympholyte-M (CEDARLANE, CL5031)
ATP solution
B27 medium
15ml conical tube
Filter mesh (pore diameter:40μm)
Pasteur pipette
Micro pipettes and their tips
Pipette aid and their tips
24 well culture dish
Swing rotor centrifuge
5% CO2 incubator
Sterilized small scissors
Methods
- Harvest spleens from 1 week-old mice
- Put spleens into a 15 ml tube and mince them well to a paste with sterilized small scissors
- Add 5.5ml of HBSS
- Suspend the spleen paste in HBSS through a Pasteur pipette
- Strain the solution through a filter mesh (pore diameter:40μm) and collect the strained solution into a new 15ml conical tube
- Add 5ml of Lympholyte-M slowly into the bottom of the conical tube
- Centrifuge the conical tube at 1500g, 20 min, RT in a swinging bucket rotor
- Carefully collect a layer of lymphocytes as per standard procedure and put cells into a new15 ml conical tube**
- Centrifuge the conical tube at 800g, 10 min, RT
- Carefully the discard the supernatant
- Add 500ml of HBSS and suspend a cell pellet using a 1000μl-pipette***
- Take out 6μl of the cell suspension for cell counting
- Add 6μl of ATP solution (At this moment, the color of HBSS changes from red to yellow)
- Incubate the cells horizontally at 37℃ (in the 5% CO2 incubator) for 15 min**** (During this time, count the number of cells)
- Centrifuge the incubated cells at 1500rpm, 5min, RT
- Carefully the discard supernatant*****
- Add B27 medium at 1×106
cells per ml
- Add 1μl of bFGF solution per 1ml to the cell suspension
- Plate the cell suspension, 1ml per well in a 24-well culture dish
- Place the culture dish into the 5% CO2 incubator and culture until cells form cell clusters (approximately for a week)
Recipes
*Sub-cultured cells or previously frozen stocked cells tend to not form cell clusters. I highly recommend the primary culture
**Contamination of red blood cells prevents cells from forming cell clusters.
***At this point, Dr. Vacanti and Dr. Kojima highly recommend to trituration of the cells with a thin-glass pipet (A). Also, I recommend using SLO (Streptolysin O) treatment if the cells don’t form clusters well (B).
(A) Trituration
- Burn a Pasteur pipette and stretch it out to make a thin-glass pipette.
- Pass the cell suspension through the thin-glass pipet for 20 min.
- Centrifuge the cells at 1200rpm for 5min at RT.
- Resuspend the cells in 494μl of HBSS
- Go back to step 13 in <Method>
(B) SLO treatment
Preparation of SLO stock solution
- Dissolve the SLO powder in water to 100μg/ml on ice
- Sterilize the SLO solution through a sterilizing filter
- Aliquot 10μl in 200μl tubes
- Store at -20℃
Methods
- Dilute the SLO stock solution 1:10 in HBSS
- Resuspend the cells in HBSS and aliquot 500,000 cells per reaction in 1.5ml tubes
- Centrifuge the cells in 1.5ml tubes (300g, 5 min, 4℃).
- Resuspend with 488μl of HBSS using a 1000μl-pipette.
- Incubate the cells at 37℃ for 2 min
- Add 12μl of diluted SLO solution
- Incubate at 37℃ for 50 min
- Collect the cell suspensions from all reaction tubes into a 15ml conical tube
- Centrifuge cells in the conical tube (400g, 10min, 4℃)
- Resuspend cells in 494μl of HBSS using a 1000μl-pipette
- Go back to step 13 in <Method>
****Try to extend the incubation time to 20 min if the cells don’t form cell clusters.
*****Remove the supernatant very carefully as if to wipe off an inner wall of the conical tube. The left-over Lympholyte solution prevent cells from forming cell clusters.
Typical Result
STAP cell cluster derived from Oct4-GFP mice
Bright field (left), Oct4-GFP (middle), Autofluorescence (right)
These photos were taken during the STAP verification experiment in Riken CDB