Cell Culture

T87 Cell Culture and Maintenance Protocol

PDF version of T87 Cell Culture Handling

NT-1 Media (1L)

  1. Add 800 ml ddH2O.
  2. Add the following:
    4.3 g MS Salt
    30 g Sucrose
    0.18 g KH2PO4
    100 µl of 10 mg/ml Thiamine stock
    220 µl of 2 mg/ml 2,4-D stock
    100 mg myo-Inositol
  3. Adjust the pH to pH 5.8 with 5M NaOH.
  4. Adjust the whole solution volume to 1 L.
  5. Distribute 75 ml media into 250 mL flasks. Cover the flasks.
  6. Autoclave the media for 20 min.

Suspension Cell Culture

The suspension cell culture was maintained on a rotary shaker (120 rpm) at 24C under constant light.
Every 7 days, transfer cells into fresh NT-1 media (1:20 v/v) for subculture.

 

Maintenance and Cryopreservation of PSB‐D and PSB‐L suspension cultures

PDF version of PSB-D and PSB-L Cell Culture Handling   Protocol tested by ABRC.

Immediately upon arrival store cells in liquid nitrogen or begin re‐initiation protocol. This is especially important for PSB‐L.


Cell suspension (CS/MSMO) medium (1 L)

4.43 g MSMO (minimal organics Sigma #M6899)
30 g Sucrose
900 ml dH2O
50 μl kinetin (1 mg/ml stock solution)
500 μl NAA (1 mg/ml stock solution)
Adjust pH to 5.7 with 1M KOH
Adjust volume to 1000 ml
Aliquot to 25 ml and 125 ml flasks
Autoclave for 20 minutes, allow to cool
For plates add 1% agar before autoclaving


Re‐initiation from cryostock

1. Thaw 1 aliquot each PSB‐D and PSB‐L in water bath at 45C for 2 minutes.
2. In laminar flow hood, pour aliquot on plate and spread with spatula. Seal with surgical tape, wrap PSB-D plate in foil and keep at 25C. Place PSB-L plate in 16 h light 8 h dark cycle at 21C.
3. In approximately 14 days, after you notice good callus growth, scrape cells from surface of agar and chop into small pieces with cell scraper. Divide cells into two 25 ml flasks containing 10 ml CS liquid medium. Wrap PSB‐D in foil and place in incubator at 25C with shaking at 130 rpm. Place PSB‐L in 16h light 8h dark cycle at 21C with shaking at 130 rpm.
4. After 7 days, sub‐culture 7 ml of small culture in 43 ml CS medium. Use each 10 ml culture to inoculate one larger culture. If they are difficult to pipette, use a sterile graduated tube to measure 7 ml. Wrap PSB‐D in foil and place in incubator at 25C with shaking at 130 rpm. Place PSB‐L in 16h light 8h dark cycle at 21C with shaking at 130 rpm.


Maintenance

Every 7 days, sub‐culture 7ml culture in 43 ml CS medium. Wrap PSB‐D in foil and place in incubator at 25C with shaking at 130 rpm. Place PSB‐L in 16h light 8h dark cycle at 21C with shaking at 130 rpm.


Cryopreservation

Pre‐freeze (PF) medium (250 ml) recipe is the same as CS except with 0.6 mol/L mannitol
1.1 g MSMO (minimal organics Sigma #M6899)
7.5 g sucrose
225 ml dH2O
12.5 μl kinetin (1mg/ml stock solution)
125 μl NAA (1mg/ml stock solution)
27 g mannitol
Adjust pH to 5.7 with 1M KOH
Adjust volume to 250 ml
Aliquot to 100 ml flasks (43 ml each)
Autoclave for 20 minutes and allow to cool

Cryoprotective medium (250 ml)
1.1 g MSMO (minimal organics Sigma #M6899)
85.6 g sucrose
200 ml dH2O
12.5 μl kinetin (1 mg/ml stock solution)
125 μl NAA (1 mg/ml stock solution)
11.5 g glycerol (use about 20 ml water to rinse out container used to weigh glycerol)
2.5 g proline
Adjust pH to 7 with 1M KOH
Filter sterilize
Add 9 ml filter sterilized DMSO

1. Sub‐culture 7 ml culture in 43 ml CS medium. Wrap PSB‐D in foil and place in incubator at 25C with shaking at 130 rpm. Place PSB‐L in 16h light 8h dark cycle at 21C with shaking at 130 rpm.
2. After 7 days, sub‐culture 10 ml culture in 40 ml PF medium. Wrap PSB‐D in foil and place in incubator at 25C with shaking at 130 rpm. Place PSB‐L in 16h light 8h dark cycle at 21C with shaking at 130 rpm. Also continue sub‐culturing in CS media as described in maintenance section to maintain a back‐up culture in case cryopreservation fails.
3. After 2 days, transfer PF cultures to 50 ml Falcon tubes and centrifuge for 1 minute at 133g
4. Aspirate off the supernatant leaving 2‐2.5 ml liquid with most of the cells in bottom of tube.
5. Add 6 ml of cryoprotective media to each tube and gently shake to re‐suspend then place in ice water for 1 hour.
6. Aliquot 2 ml of cells in cryoprotective media to cold 2 ml Nalgene tubes (store tubes in fridge before use and keep on ice while aliquotting).
7. Place tubes in Mr Frosty filled with isopropanol that has been cooled in fridge.
8. Place Mr Frosty on bottom shelf of ‐80C freezer for approximately 1 hour 40 min, follow the temperature using an electronic thermometer until it reaches ‐50C
9. Transfer tubes to liquid nitrogen storage.

For Agrobacterium‐mediated transformation please refer to the protocol from VIB [pdf].

 

Black Mexican Sweet (BMS) Cell Culture and Maintenance Protocol

PDF version of BMS Cell Culture Handling

M237 Media (1L)

  1. Add 900 ml ddH2O.
  2. Add the following:
    4.3 g MS Salt
    30 g Sucrose
  3. Adjust pH to 5.6 with 1M KOH
  4. Adjust volume to 1L
  5. Distribute 50 ml media into 250 mL flasks. Cover the flasks.
  6. Autoclave for 20 min

Let the media cool down (approx. 60°C), and add 1 mL MS vitamins (Sigma-Aldrich, cat# M3900-50ML) + 0.5ml 2,4-D (2mg/ml, filter-sterilized).
 
For plates add 0.7% of agar before autoclaving.

Suspension Cell Culture

The suspension cell culture was maintained on a rotary shaker (120 rpm) at 24C in dark conditions. Every 3 weeks, transfer cells into fresh M237 media (1:10 v/v) for subculture.

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