Human scalp skin biopsies were acquired as discarded tissue from surgical procedures following informed consent using IC-REC approved consent forms. Isolation of DP and PFI cells from biopsies was performed using a micro-dissection technique as previously reported [49 (link)]. Growth media (GM) consisted of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S, Thermo Fisher, 15070-063). When osteogenic media (OM) was implemented, it consisted of low glucose DMEM (LG-DMEM, Thermo Fisher, 31885-023) containing 10% FBS and 1% P/S, and further supplemented with 100 nM dexamethasone (Sigma Aldrich, D4902), 50 μM l -ascorbic acid 2-phosphate (Sigma Aldrich, A8960) and 10 mM β-glycerol phosphate (Sigma Aldrich, G9422). Adipogenic media (AM) consisted of DMEM, 15% FBS, 1% P/S, 100 nM dexamethasone, 2.07 μM insulin (Sigma Aldrich, I9278), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma Aldrich, I7018), and 200 μM indomethacin (Sigma Aldrich, I7378). PFi were used from matched patient biopsies as the DP above. Rat DP were isolated from rat whiskers using the same technique [49 (link)], while human BM-MSC were purchased from Merck Millipore (SCC034).
I7018
Manufactured by Merck Group
Sourced in Sweden, China, United States
The I7018 is a laboratory instrument designed for spectrophotometric analysis. It is a compact and versatile device that measures the absorption or transmittance of light through a sample, enabling the quantification of various chemical and biological analytes.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (6 mentions)
Insulin, by Merck Group (4 mentions)
I5500, by Merck Group (4 mentions)
D4902, by Merck Group (3 mentions)
I9278, by Merck Group (3 mentions)
Isobutylmethylxanthine, by Merck Group (3 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (2 mentions)
Rosiglitazone, by Merck Group (2 mentions)
I0546, by Merck Group (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
6 well cell culture plate, by Corning (2 mentions)
I8830, by Solarbio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem lg, by Thermo Fisher Scientific (1 mentions)
A8960, by Merck Group (1 mentions)
I7378, by Merck Group (1 mentions)
Scc034, by Merck Group (1 mentions)
G9422, by Merck Group (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
R2408, by Merck Group (1 mentions)
14 protocols using i7018
1
Isolation and Culture of Dermal Papilla and Papilla-Derived Fibroblasts
2
Plasma cGMP Measurement Protocol
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To prevent cyclic guanosine monophosphate (cGMP) degradation, an inhibitor of cAMP/cGMP phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine, 10 uM (I7018; Sigma-Aldrich, Merck, Sweden) was added to the plasma collected for cGMP measurement. Plasma cGMP concentration was analyzed using a commercially available ELISA (Cayman Chemicals #581021 BioNordika, Solna, Sweden), according to the manufacturer’s instructions.
3
Isolation and Differentiation of Mouse Adipocytes
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Mouse subcutaneous inguinal fat pads were removed and washed with PBS pH7.4 (Gibco) and then minced. The minced tissue was digested for 15–20 min at 37°C in the digestion buffer (10 mM CaCl2, 2.5 unit/ml collagenase D, 2.4 units/ml Dispase II in PBS). The digested tissue was filtered through a 100 μm mesh and centrifuged at 600 × G for 5 min. Floating adipocyte fraction was removed. The resulting pellets were resuspended and further filtered through the 40 μm nylon cell strainers (BD Biosciences). Stromal vascular fraction (SVF) cells were maintained in DMEM/F12 GlutaMAX (Invitrogen) containing 15%FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For differentiation, confluent preadipocytes were treated with medium containing 15% FBS, 0.5 mM isobutylmethylxanthine (I7018; Sigma), 1 μM dexamethasone (D4902; Sigma), 2 μg/mL insulin (I0546; Sigma), and 1 μM rosiglitazone (R2408; Sigma) for 48 h. Adipocytes were then maintained in medium containing 15% FBS and 2 μg/mL insulin. After 6–7 days of induction, differentiated cells were challenged with 5 or 20 μM Yoda1 (SML1558, Sigma), 1 μM TAK-242 (5.08336, Millipore) and 5 μM GsMTx-4 (STG-100, Alomone) or vehicle for 18–20 h. Cells were collected for further analysis.
4
3T3-L1 Adipocyte Differentiation Protocol
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Pre-adipocytes 3T3-L1 were cultured in high glucose Dulbecco’s modified Eagle’s medium (HyClone) with 10% FBS and 1% penicillin streptomycin. Cell differentiation was induced by adding cell culture medium I (DMEM with 10% FBS, 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine (I7018, Sigma, Shanghai, China), 1 µM dexamethasone (D4902, Sigma, Shanghai, China) and 10 µg/mL insulin (I9278, Sigma, Shanghai, China)) for two days. Then, they continued to culture in cell culture medium II (DMEM with 10% FBS, 1% P/S and 10 µg/mL insulin) for another two days. Then cell culture medium (DMEM with 10% FBS and 1% P/S) was changed every two days for two times according to the method we described earlier [21 (link)].
5
3T3-L1 Cell Differentiation Protocol
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3T3-L1 cells isolated from male mouse embryos were cultured in DMEM (11965092, Gibco) with 10% newborn calf serum (16010159, Gibco) and they were differentiated in DMEM containing 10% fetal bovine serum (FBS, 26140079, Gibco), 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX, I7018, Sigma-Aldrich), 0.25 lM dexamethasone (D4902m, Sigma-Aldrich), and 1 μg/ml insulin (I5500, Sigma-Aldrich). Before the experiments, both 3T3-L1 undifferentiated and differentiated cells were briefly serum-starved in DMEM-0.5% newborn calf serum or fetal bovine serum for 8 hours.
6
Preadipocyte Differentiation Protocol
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Primary preadipocytes were seeded in 6-well cell culture plates (Corning Costar Corp., Cambridge, MA, USA) and cultured in BCM. After cells were ~70% confluent, the BCM was replaced by freshly-prepared differentiation culture medium 1 (DCM1) adding 0.5 mM 3-Isobutyl-1-methylxanthin (IBMX; I-7018; Sigma-Aldrich), 1 μM dexamethasone (D-4902; Sigma-Aldrich) and 1 μg/mL insulin (I-5500; Sigma-Aldrich) in BCM to induce preadipocytes differentiation. After 2 days, DCM1 was replaced with differentiation culture medium 2 (DCM2), which contained a final concentration of 1 μg/mL insulin in BCM, to maintain the differentiation state. Fresh DCM2 was replaced every other day for about 10 days until visible lipid droplets appeared in the cell, indicating that cells had completed differentiation. After differentiation, the number of mature adipocytes was 4.0 × 105 per 6-well plate.
7
Adipocyte Differentiation and Zinc Treatment
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Cell culture and adipose differentiation were performed as previously reported [26 (link)]. Briefly, 3T3-L1 preadipocytes (CL-173, American Type Culture Collection, Manassas, VA, USA) were cultured in basic medium (DMEM medium (11995040, Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (10099-141, Gibco, Shanghai, China), 100 U/mL penicillin and 100 µg/mL streptomycin (10378016, Gibco) at 37 °C and 5% CO2. Once 75% confluence was reached, cells were subcultured into 12-well plate at 90% confluence. 2 days after 100% confluence, cells were treated with 1 µg/mL insulin (I5500, Sigma, Shanghai, China), 1 µM dexamethasone (D1881, Sigma, Shanghai, China) and 0.5 mM isobutyl methyl xanthine (IBMX) (I7018, Sigma, Shanghai, China) for 3 days. Cells were then maintained in 1 µg/mL insulin-supplemented basic medium. Medium was freshly changed 3 times in the next days. On day 9, fully differentiated adipocytes were washed 3 times with serum-free DMEM, followed by incubating in serum-free DMEM medium for 14 h. Cells were then treated with 50 µM zinc or the vehicle for 6 h.
8
Trafficking of Aquaporin 2 in Cysts
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Fully formed cysts were washed and incubated in serum free medium 120 min prior to treatment. To stimulate the trafficking of AQP2, 1x10-8 M AVP and/or 1x10-5 M FK or CPT-cAMP (8-(4-Chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate) was added to the medium for 30 minutes. For “cysts” treated with CPT-cAMP, a preincubation with 3-isobutyl-1-methylxanthine (IBMX) (Sigma Aldrich I7018) at a final concentration of 1mM for 30 min was performed prior to adding cAMP (Sigma Aldrich C8988). Treating cells with VP and FK alone or in combination gives similar result.
9
Isolation and Differentiation of Murine White Adipose Stromal Vascular Fraction Cells
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Primary white adipose SVF cells were cultured as we described previously (Shan et al., 2016 (link)). Briefly, the inguinal fat pad was collected from 6-week-old female mice and washed with PBS twice. Then, the fat pad was minced with scissors and digested with collagenase type I (1.5 mg/ml, #SCR103, Sigma-Aldrich) at 37°C for 40 min. When the digestion was finished, the growth medium contained 85% high glucose DMEM medium (#11965126, Thermo Fisher Scientific) and 15% fetal bovine serum (#10099141, Thermo Fisher Scientific) was added to dilute the collagenase. The tissue debris was removed through a 70-μm cell strainer. The medium was subjected to centrifuge to get SVF cells pellet. SVF cells were resuspended with the growth medium. When the cells reached 90% confluence, they were induced to adipogenesis, with a cocktail containing DMEM, 10% fetal bovine serum, 2.85 mM recombinant human insulin (#I8830, Solarbio), 0.3 mM dexamethasone (#D8040, Solarbio), and 0.63 mM 3-isobutyl-methylxanthine (#I7018, Sigma-Aldrich). After 4 days, the cocktail was switched to a DMEM medium supplemented with 10% fetal bovine serum, 10 nM triiodothyronine (T3, #T6397, Sigma-Aldrich), and 200 nM insulin to induce mature adipocytes.
10
Adipocyte Differentiation and siRNA Delivery
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