— Dan Yamamoto-Evans 2019/11/30 14:17

• FBS
• RPMI‐1640 medium
• x100 Penicillin-Streptoadivin
• Cell stock

• Warm the FBS (50mL, stored at -20˚C) and RPMI-1640 (500mL, stored at 4˚C) in the water bath @37˚C.
• Thaw x100 Pen-strep
• Add 50 mL of FBS to RPMI-1640 (Final 10%)
• Add 5 mL of x100 Pen-strep to RPMI-1640 (Final x1)
• Label the bottle, mix and warm in the water bath.

• Take out the cell-stock from liquid nitrogen tank, and put on ice.

• Aliquot 5 mL of the warm media to a new falcon tube.
• Thaw the cell-tube on the water bath, and once it is thawed, add the entire sample to the aliquotted medium.

• Spin down sample at 1000 rpm for 5 min.
• Remove sup.
• Add 10 mL of the same medium to the tube, and resuspend by pippette to mix.
• Take out an 10cm dish, and label it.

• Add the sample to dish, and spread cells by moving plate horizontally and than vertically.
• Incubate in the human cell culture incubator.

• Clean the spcace with EtOH before/after use.
• DO NOT put lid from reagents etc on the surfaces.
• EtOH stuff, but NOT pippettes and plates.
• Put back the medium in Human medium fridge.

— Dan Yamamoto-Evans 2019/11/30 15:54