Protocols
&
Resources
Software & repositories
https://github.com/Svensson-Lab
AlphaFold ligand-receptor binding prediction
These scripts will predict ligand-receptor interactions using AlphaFold version 2.3.1.
Prohormone Predictor - this program will annotate all proteins predicted to generate peptides generated by proteolytic cleavage by the PCSK enzymes.
A web app for determining the subcellular location of proteins (by UniProtID).
Scripts to run phosphoproteomic analyses.
Plasmids & reagents
Svensson Lab plasmids can be found at Addgene.
We have made the C57BL/6-Ism1-flox mice (#036776) available at JAX.
For other plasmids, proteins or reagents, please email katrinjs(at)stanford.edu
Protocols
Large scale recombinant secreted protein production in mammalian cells
Day −1: Culture
Culture Expi293F cells: #A14527 (ThermoFisher) in 125-mL polycarbonate, disposable, sterile, vent-cap Erlenmeyer shaker flasks containing 30 mL of Expi293 Expression Medium. Incubate the cells in a 37°C incubator with ≥ 80% relative humidity and 8 % CO2 on an orbital shaker platform.
The day prior to transfection (Day −1), split the Expi293F culture to a final density of 2.5 –3 × 10^6 viable cells/mL and grow overnight. Viability should be 95–99% to proceed with transfection.
Day 0: Transfection
Transfect cells with the ExpiFectamine 293 + endotoxin-free plasmid DNA complexes according to the instructions (#A14527 (ThermoFisher). After 4-5 days, collect the conditioned media and proceed with protein purification.
Day 4-6: Protein purification
For manual purification, use His GraviTrap TALON cobalt column (GE29-0005-94) described here for a protein yield of 15 mg per purification. When scaling up, use HisPur™ Ni-NTA Chromatography Cartridges #90099 with FPLC.
Buffers:
1. Binding buffer: 50 mM sodium phosphate + 300 mM NaCl, pH 8. Filter using < 0.22um.
2. Wash buffer: 50 mM sodium phosphate + 300 mM NaCl + 5 mM imidazole, pH 8
3. Elution buffer: 50 mM sodium phosphate + 300 mM NaCl+ 200 mM imidazole, pH 8
Spin the conditioned media twice at 400 x g for 10 min to remove cell debris.
Filter the media using a 0.2 μm filter and adjust the pH of the media to 8.
Cut off the bottom tip of the His GraviTrap column, remove the top cap and empty the column from the storage buffer
Add 10 ml binding buffer to the column
Add your sample to the column.
Wash with 10 ml binding buffer
Wash with 10 ml washing buffer
Add 2 ml elution buffer 3X to the column and collect the eluate containing the purified protein.
Perform buffer exchange to PBS using dialysis cassette.
Measure the protein concentration.
Aliquot the protein and store in -80°C. Typical concentration is 1-2mg/ml.
Perform quality controls on the recombinant proteins
SDS page followed by silverstain
Western blot using an anti-his antibody and an antibody to the protein of interest
Endotoxin test - the level should be < 0.1 U/mg protein
Unbiased proteomics to exclude potential contaminating proteins
Bioactivity test using a cellular or enzymatic assay
Preadipocyte isolation and differentiation protocol
(Svensson et al, 2016, Jiang et al 2021)
Differentiation components
Rosiglitazone [1μM]: (Cayman; 71740)
Isobutylmethylxanthine (IBMX) [0.5 mM]
Dexametasone [1μM]: (Sigma; D4902)
Insulin [5μg/ml]
WAT isolation buffer
For 10 ml solution (sufficient for 10 fat pads from mice):
10 ml PBS
11.1 mg CaCl2 - final conc 10 mM
48 mg dispase II - final conc 2.4 U/ml dispase II (04942078001, Roche)
•100 mg dispase D - final conc 10 mg/ml collagenase D (11088858001, Roche)
Components needed for cell culture
DMEM/F12 + Glutamax media: (Invitrogen; 10565-042)
FBS: Fisherbrand™ Research Grade Fetal Bovine Serum
Primocin: (Invivogen; ant-pm2) – Use 1ml/500ml media
Trypsin EDTA 1X: 0.25% (Thermo-Fisher, 25200-056)
Collagen-I-coated plates
iWAT Growth media (used from time of isolation)
90% DMEM + 10% FBS + Penicillin-streptomycin + Primocin Note: Remove primocin at day 0 of differentiation
iWAT Differentiation cocktail (used from day 0- day 2)
5 μg/ml Insulin
1 μM Rosiglitazone
0.5 mM IBMX
1 μM Dexamethasone
iWAT Maintenance cocktail (used from day 2 - day 4)
5 μg/ml Insulin
1 μM Rosiglitazone
Isolation of stromal vascular cells (SVF)
1. Mice: C57/b6 mice, 4 weeks old, inguinal (subcutaneous) fat pad. From 5 mice, isolate 10 inguinal fat pads and place in a 5 ml eppendorf tube
2. Mechanically digest the tissue for 5-10 min using spring scissors.
3. Add WAT isolation buffer and incubate at 37C for 30-45 min. Invert tubes every 10 mins.
Note: Collagenase lots can vary in enzymatic activity. Pay attention to the digestion process when using a new lot and adjust digestion time as needed to avoid overdigestion.
4. Add 10 ml of growth medium, filter through a 100 μm filter and spin down at 600g for 10 min.
5. Resuspend pellet in 10 ml of growth medium, filter through a 40 μm filter and spin down at 600g for 10 min.
6. Resuspend pellet in 10 ml growth medium and plate on collagen-I-coated 10 cm plates.
7. Next day, wash cells with PBS 2-10 times and add new growth media.
8. Within 3-5 days, split to 3 10cm plates. Change media every 2 days.
9. Within 3-5 days, when cells are confluent, split to 10 10cm plates.
10. When confluent, cells are frozen down (one 10cm plate per vial, approx. 3-5 million cells) for further use.
Thawing and cell seeding
1. Thaw cells from one vial rapidly at 37C and resuspend in growth medium in a 10cm collagen-I coated plate. Expected cell viability is 80-90 %. Incubator is set at 37C, 10% CO2.
2. Change growth media on cells every day or every other day. Seed into final format when cells are 100 % confluent.
Seeding into final format
1. Trypsinize cells in 0.25 mM trypsin. Do not over-trypsinize.
2. Seed cells into appropriate plate format. For a 12-well, seed 100.000 cells/well on collagen-coated plates. Typically, 1 x 10cm confluent dish will be sufficient for 4 x 12-well collagen-coated plates.
3. Let cells grow until confluency (2-4 days). At this point, change media and wait another 2 days.
Differentiation
1. Day 0: Add freshly made growth media with differentiation cocktail.
2. Day 2: Change to growth media with differentiation cocktail, do not wash with PBS.
3. Day 4: Change to growth medium, do not wash with PBS.
4. Day 6: Change to growth medium, do not wash with PBS.
5. Lipid droplets should be seen around day 4 and by day 5-6 most cells will be mature.
Treatments
1. At day 6-8, treat cells with recombinant protein or activators for 30 min - 24h depending on the downstream assay. Depending on the assay, cells might be starved in serum free media overnight before stimulation.
2. Assay cells for activation of thermogenesis by gene expression, activation of intracellular signaling by western blot, or cAMP activation.
3. Always include positive treatment controls such as norepinephrine, forskolin or FGF21 recombinant protein as stimulators.
4. When performing qpcr analysis, include genes involved in differentiation and thermogenesis such as adiponectin, pparg, ap2.
Hepatocyte isolation and culture
De novo lipogenesis assay
(Jiang et al, 2021) (adapted from :Akie, T. E., & Cooper, M. P. (2015). Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes. Journal of visualized experiments : JoVE, (102), e52982. doi:10.3791/52982)
20 x radiolabeled acetate mix (200 μM cold acetate + 20 μCi radioactive 3H-acetate)
To make 1 mL 20 x acetate mix (enough for 20 wells), mix 1 mL 200 μM cold acetate + 4 μL 3H-acetate from the stock (10 mCi/mL 3H-acetate).
Measuring acetate incorporation into lipids
1. Seed cells at 50 % confluence in 12-well plates. When seeding to 12-well plates for assay, insulin should be removed 1 day before the assay.
2. Change to Serum-free Medium and add treatments for 4h-24h. For example, add 100 nM Insulin (an activator of lipogenesis) +/- different conc. of recombinant proteins of interest. Use 100 nM BSA in PBS as a negative control protein.
3. Add 50 μl of the 20x acetate mix to each well. (final conc. 10 μM cold acetate and 2 μCi 3H-Acetate).
4. Incubate for 2-4 hr. Note: optimal incubation time is cell-type dependent.
5. After 2-4h, wash the cells with PBS twice.
6. Lyse the cells with 120-200 μl of 0.1 N HCl.
7. Measure the protein concentration of each sample.
8. Extract lipids by adding 500μl of chloroform/methanol (2:1, v/v). Vortex briefly and incubate the sample at room temperature for 5 minutes.
9. Add 250 μl water and vortex. Incubate the sample at room temperature for 5 minutes.
10. Centrifuge the samples for 10 minutes at 3000g at room temperature.
11. Carefully transfer 250 μL of the lower phase to 3.5 ml liquid scintillation buffer in a scintillation vial and measure 3H activity.
12. Normalize lipid incorporation by protein amount or cell number.