Starvation of cells and re-stimulation with amino acids

Maria Manifava, Babraham Institute, Cambridge UK

Background

We have been using a protocol of nutrient starvation that induces rapid autophagy in a variety of common cell lines such as HeLa, HEK-293, HAP-1, MEFs etc. In this protocol the normal medium is replaced with a salt solution lacking amino acids, growth factors and vitamins but containing some glucose and BSA or dialysed serum. Because the morphology of cells in this starvation solution appears slightly different than in normal growth medium, we have also created another solution that contains amino acids and which does not induce autophagy but still changes cell morphology along the same lines as the starvation solution. [In general, incubation/growth of all cells in simple salt solutions makes them flatter and slightly more transparent]. In our recent paper examining mTOR dynamics in response to amino acid depletion and re-addition, we have used these solutions for both live imaging and biochemical studies. We note that these protocols are based on the pioneering work from the Sabatini lab; the difference is that we don’t start the experiment with a serum starvation overnight before the amino acid starvation step (as is done in the Sabatini protocols) but rather combine serum- and amino acid starvation in one 60 min pre-treatment. One cell line that we have used (HAP-1) required some additional components during re-stimulation and in the following protocol we will note this variation.

For starvation (both HEK-293 and HAP-1),

Cells were washed twice with pre-warmed starvation medium (140 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Glucose, 20 mM Hepes, 5 mM KCl, pH 7.4) containing either 1% BSA or 1% dialysed FBS
Cells were incubated in this medium for 60 min at 37 ^oC in tissue culture incubator.
At the end of 60 min, the medium for HEK-293 cells was replaced with new starvation medium containing 1.5X solution of MEM amino acid solution (50X stock, containing the following amino acids in one letter code R, C, H, I, L, K, M, F, T, W, Y, V, and sold by LifeTechnologies),
Cells were incubated in this solution for the indicated times, typically 10’, 20’, 30’, 45’, 60’, 75’, 90’.
For HAP-1 cells, the starvation medium was replaced with new starvation medium containing 1 mM glutamine, MEM amino acids as above, 2X solution of NE amino acids (100X stock, containing the following amino acids in one letter code G, A, N, D, E, P, S and sold by LifeTechnologies), insulin (1:500 dilution of liquid supplement at 9–11 mg/ml sold by Sigma-Aldrich) and EGF (20 ng/ml).
In some experiments using a fluorescent amino acid analogue, we also added 0.4 mM arginine to the above solution

Extended Protocol:

Pre-warm the starvation medium at 37°C and wash twice the cells (2ml/6well, or adjust accordingly). The washes are quick, and involve just the change of medium. The aim of the wash is to ensure that no amino acids have remained from the DMEM.
Incubate the cells with fresh starvation medium for 1 hr in the incubator.
During this incubation, prepare the recovery solution.
At the end of the incubation period aspirate the starvation medium, and add the recovery solution for the appropriate time points (2mls/ 6well, or adjust accordingly).
Fix or lyse the cells according to the output assay.

Solutions:

1. Starvation solution: 140 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Glucose, 20 mM Hepes, 5 mM KCl, pH 7.4. More specifically:

28 ml from 5 M NaCl, 1 ml from 1 M CaCl₂, 1ml from 1 M MgCl₂, 5.5 ml from 1 M Glucose, 20 ml from 1 M Hepes pH 7.4 (1M Hepes: 260.29gr, 1L H2O).

Add 1% Bovine Serum Albumin (heat shock fraction, pH 7, ≥98% purity) for HEK-293 cells or 1% Dialyzed Fetal Bovine Serum (Gibco 26400044) for HAP-1 cells.

2. Recovery solution for HEK-293 cells: starvation solution plus 1.5x MEM Amino Acids Solution (50X stock):

For 50 ml solution, 48.5 ml starvation solution, 1.5ml MEM amino acids.

Very important: Bring up the pH to 7.4 (Initial pH is 6.4. Add 50 ml, approximately, of 8M NaOH)

3. Recovery solution for HAP-1 cells: starvation solution plus, 2X MEM, 2X MEM-NEAA, 4mM Glutamine, 200 μg/ml EGF, 1:500 Insulin 9.5-11.5 mg/ml.

For 10ml solution: 9.06 ml starvation solution, 400 ml MEM amino acids, 200 ml MEM-NEAA, 200 ml glutamine.

Bring up the pH to 7.4 (Initial pH is 3.8. Add 30 ml, approximately, of 8M NaOH).

Finally add 20 ml EGF (10 μg/ml) and 20 ml Insulin (10 mg/ml).

For both recovery solutions: Pre-warm for only a short period, to retain the maximum activity of the amino acids and the growth factors.

Reagent	Company	Catalogue number	Web-page	Notes
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	11140	https://www.thermofisher.com/order/catalog/product/11140050#/11140050	Upon receipt, make small aliquots. Store at -20°C. Thaw before use. Use each aliquot once
MEM Amino Acids Solution (50X) liquid	Thermo Fisher Scientific	11130036	https://www.thermofisher.com/order/catalog/product/11130036#/11130036	Upon receipt, make small aliquots. Store at -20°C. Thaw before use. Use each aliquot once.
Epidermal Growth Factor, human (hEGF), 5 mg	Sigma	E9644	https://www.sigmaaldrich.com/catalog/product/sigma/e9644?lang=en&region=GB	Stock: Dilute stock of 200 μg/ml to 10 μg/ml in 10% acetic acid plus 0.1% BSA fatty acid free. Store at -20°C in small aliquots.
Insulin solution from bovine pancreas	Sigma	I0516	https://www.sigmaaldrich.com/catalog/product/sigma/i0516?lang=en&region=GB	10 mg/mL insulin in 25 mM HEPES, pH 8.2, Bio Reagent, sterile-filtered, suitable for cell culture.
L-Glutamine (200 mM)	Thermo Fisher Scientific	25030149	https://www.thermofisher.com/order/catalog/product/25030149#/25030149	Upon receipt, make small aliquots. Store at -20°C. Thaw before use. Use each aliquot once.
Fetal Bovine Serum, dialyzed, US origin	Thermo Fisher Scientific	26400044	https://www.thermofisher.com/order/catalog/product/26400044#/26400044

Equipment:

Small, heated water bath (37 °C) (Grant instruments Ltd, JB Nova Unstirred Water Bath)
Humidified incubator: 37 °C, 5% CO₂(CO₂ incubator, Panasonic, catalog number: MCO-170AI )
Bibby Vortexer (HC 502)
WPA PH-meter (CD500)
Sartorius Excellence (E1200S) balance
Envair Bio2+ Class II Microbiological Safety Cabinet
Sanyo MEDICOOL fridge (MPR 513)
Liebherr ProfLine Freezer (MPR 161DCH)
Drummond Pipet Aid ( 4-000-220-TCE)

How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Dynamics of mTORC1 activation in response to amino acids.

Maria Manifava , Matthew Smith, Sergio Rotondo , Simon Walker, Izabella Niewczas , Roberto Zoncu , Jonathan Clark , Nicholas T Ktistakis eLIFE DOI: 10.7554/eLife.19960