TITLE: SH-SY5Y culturing
AUTHORS: Laura  Smith, David  C

[DESCRIPTION]
SH-SY5Y neuroblastoma cells were cultured in a 1:1 mixture of Ham’s F-12 and DMEM growth medium supplemented with 10% FBS, non-essential amino acids (NEAA: 0.1 mM of: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine) and penicillin/streptomycin antibiotic cocktail (50ng/ml) at 37°C and 5% CO2. Cells were passaged at around 80% confluency using trypsin digestion. Culture medium was removed from cells, centrifuged at 1000 RPM for 5 minutes to remove floating cells and stored at 80°C for analysis. Cells were washed once in PBS and trypsin was added (0.25% trypsin in Versene). Following an incubation of 2 minutes, the plates were tapped to detach cells. The trypsin was neutralised in 9 volumes of normal growth medium. For continued culturing, a fraction of cells was transferred to a falcon tube and pelleted for 10 minutes at 1000 RPM (200 x g). Pellets were resuspended in the appropriate volume of normal medium and split into new plates. If required, cells were counted prior to seeding using the C-Chip NanoEnTek DHC-N01 counter. Cell line stocks were generated by resuspending cell pellets in 1 mL of freezing medium (10% DMSO in FBS). 

[STEPS]
SECTION: Characteristics
1. ATCC #      CRL-2266
Human neuroblastoma      cell line, cloned from SH-SY5, which is from SH-SY, which is from SK-N-SH.      The original cell line was isolated      from a woman's metastatic bone tumor in 1970.
Known      to be dopamine beta hydroxylase active, acetylcholinergic, glutamatergic      and adenosinergic.
Grown      as adherent cells; dividing cells can form clusters of cells which are      reminders of their cancerous nature, but certain treatments such as      retinoic acid and BDNF can force the cells to dendrify and differentiate.
Doubling      time = 48h
SECTION: Complete growth medium
2. 1:1      mixture of
- F12 (LT # 31765-068, 1802 mg/L glucose and 110 mg/ml sodium pyruvate) and,
- DMEM (# 61965-059; 4500 mg/L and no pyruvate) both Glutamax medium
Or DMEM/F12 (#31331028)
Fetal      bovine serum (FBS, 10% final) – 1 / 10 dilution
Pen / Strep     (LT # 15140-122, 50 U/ml and 50 ng/ml final) -- 1      / 200 dilution
MEM / NEAA     (LT # 11140-035) -- 1 / 100 dilution
As      required -- Sodium pyruvate (Sigma #      S8636, 1 mM final) – 1 / 100 dilution
As      required -- Uridine (Sigma # U3003, stock      of 25 mg/ml in H2O filter-sterilised; 50 ug/ml final) – 1 / 500 dilution
- Omit uridine and pyruvate to select against ro cells (selective medium)
- uridine not necessary for general cell growth but helps MRC defective cells
As      required, as permitted by DSO -- Fungizone     (LT # 15290-026; original of 250 ug/ml) – 1 /      100 dilution
SECTION: Subculturing by trypsinisation, and cell counting
3. Wash      cells grown onto 10 cm Ø plate with PBS.
Add 1      ml diluted trypsin (4 ml of 2.5%, LT #      15090-046, into 100 ml Versene LT #      15040-033) and leave at R/T for several mins.
Tap      the plates and add 2 ml complete medium to stop trypsination.Pipette trypsinised cells to break down      clumps of cells, triturate using 21G needles if necessary.
To a      50 ul sample, add equal vol of 0.4% Trypan blue (Sigma # T8154 or LT #      15250-061); fed it to a hemacytometer.
Optional      -- to pellet cells and resuspend into fresh medium.
Replate      the cells accordingly; typically 8 ml medium is used for 10 cm Ø plate.
Subcultivation      ratio of 1:4 to 1:16 is recommended; medium renewal every 2-3d.
SECTION: Freezing and thawing
4. 1.After centrifugation to pellet cells, resuspend in Freezing medium (90% FBS / 10% DMSO filter sterilised) and aliquot into Freezing vials; typically a confluent 10 cm Ø plate of ~ 6 million cells in 1ml.Freeze cells in a stepwise fashion from -80’C to liq N.
2.Thaw cells at 37’C.