Preparation of Reagents

Preparation of Reagents

ATP solution

Adenosin5’-triphosphate disodium salt hydrate (Sigma, A3377)

Water (Sigma, w3500, RNBC5693)

1. Dilute 110.57mg of ATP into 1ml of H2O（200nM in H2O）
2. Sterilize the ATP solution through a sterilizing filter
3. Aliquot in 30μl increments
4. Store at -20℃

bFGF solution

bFGF (WAKO, 060-04543, 100μg)

PBS (Gibco, 14170)

1. Dilute 100μg of bFGF into 100μl of PBS
2. Aliquot in 10μl increments
3. Store at -20℃

B27 medium

F12/DMEM 1:1 (Gibco, 11330)

B27 (Gibco, 17504)

LIF (Millipore, ESGRO, mLIF, ESG1107)

1. Add 10ml of B27 and 50μl of LIF into 500ml of F12/DMEM medium
2. Sterilize the mixture through a sterilizing filter
3. 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

1. Harvest spleens from 1 week-old mice
2. Put spleens into a 15 ml tube and mince them well to a paste with sterilized small scissors
3. Add 5.5ml of HBSS
4. Suspend the spleen paste in HBSS through a Pasteur pipette
5. Strain the solution through a filter mesh (pore diameter:40μm) and collect the strained solution into a new 15ml conical tube
6. Add 5ml of Lympholyte-M slowly into the bottom of the conical tube
7. Centrifuge the conical tube at 1500g, 20 min, RT in a swinging bucket rotor
8. Carefully collect a layer of lymphocytes as per standard procedure and put cells into a new15 ml conical tube**
9. Centrifuge the conical tube at 800g, 10 min, RT
10. Carefully the discard the supernatant
11. Add 500ml of HBSS and suspend a cell pellet using a 1000μl-pipette***
12. Take out 6μl of the cell suspension for cell counting
13. Add 6μl of ATP solution (At this moment, the color of HBSS changes from red to yellow)
14. Incubate the cells horizontally at 37℃ (in the 5% CO2 incubator) for 15 min**** (During this time, count the number of cells)
15. Centrifuge the incubated cells at 1500rpm, 5min, RT
16. Carefully the discard supernatant*****
17. Add B27 medium at 1×106 cells per ml
18. Add 1μl of bFGF solution per 1ml to the cell suspension
19. Plate the cell suspension, 1ml per well in a 24-well culture dish
20. Place the culture dish into the 5% CO2 incubator and culture until cells form cell clusters (approximately for a week)

Recipes

*Liver would be easier than other tissues. At the verification experiment of STAP, Dr. Niwa’s group independently succeeded the recreation of cell clusters which expressed pluripotent stem cell markers such as Oct4 and Nanog from liver cells (seehttp://biorxiv.org/content/biorxiv/early/2015/09/28/027730.full.pdf).

*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

1. Burn a Pasteur pipette and stretch it out to make a thin-glass pipette.
2. Pass the cell suspension through the thin-glass pipet for 20 min.
3. Centrifuge the cells at 1200rpm for 5min at RT.
4. Resuspend the cells in 494μl of HBSS
5. Go back to step 13 in <Method>

(B) SLO treatment

(Reference: “Permeabilization of donor cells” at www.collaslab.net)

Preparation of SLO stock solution

1. Dissolve the SLO powder in water to 100μg/ml on ice
2. Sterilize the SLO solution through a sterilizing filter
3. Aliquot 10μl in 200μl tubes
4. Store at -20℃

Methods

1. Dilute the SLO stock solution 1:10 in HBSS
2. Resuspend the cells in HBSS and aliquot 500,000 cells per reaction in 1.5ml tubes
3. Centrifuge the cells in 1.5ml tubes (300g, 5 min, 4℃).
4. Resuspend with 488μl of HBSS using a 1000μl-pipette.
5. Incubate the cells at 37℃ for 2 min
6. Add 12μl of diluted SLO solution
7. Incubate at 37℃ for 50 min
8. Collect the cell suspensions from all reaction tubes into a 15ml conical tube
9. Centrifuge cells in the conical tube (400g, 10min, 4℃)
10. Resuspend cells in 494μl of HBSS using a 1000μl-pipette
11. 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