20.109(F19): Prep notes for M3

M3D1

24 hr preceding laboratory:

• seed MEF (250K) in T25 flasks, 1 per team
• cell expansion schedule for D1 and D2 prep

in tissue culture room (per team):

• 1 fresh T75 flask
• 3.5 mL PBS
• 1.5 mL trypsin
• 15 mL media
• not aliquoted: trypan blue

in main lab (per team):

• none

M3D2

48 hr preceding laboratory:

• seed MEF (2-3M) in T75 flasks, 4 per team

in tissue culture room (per team):

• 25 mL PBS
• 25 mL serum-free media
• not aliquoted: ligands

in main lab (per team):

• 15 mL PBS
• 5 mL trypsin
• 15 media
• 1.5 mL PBS containing protease inhibitor
• not aliquoted: protease inhibitor

M3D3

in tissue culture room (per team):

• none

in main lab (per team):

• 75 μL Laemmli sample buffer
• 30 mL blocking buffer
• not aliquoted: molecular weight standards, TGS running buffer, dH₂O

M3D4

in tissue culture room (per team):

• none

in main lab (per team):

• 10 mL blocking buffer (2 per team)
• not aliquoted: TBS-T

M3D5

in tissue culture room (per team):

• none

in main lab (per team):

• none