hMSC-laden hydrogels (n = 3) were prepared as described above, except the cell suspension was washed extensively with “heavy” SILAC medium (SILAC αMEM, no nucleosides, no lipoic acid prepared in-house and supplemented with 0.1 mg/mL heavy isotopically-labeled lysine and arginine, #CNLM-291-H, #CNLM-539-H, Cambridge Isotope Laboratories, Inc.) prior to encapsulation. LD and HD hydrogel cultures were cultured for 72 h in “heavy” SILAC medium with 10% dialyzed FBS (against PBS using Amicon 3K MWCO units, Millipore) and 1% antibiotic-antimycotic solution. Samples were decellularized with 0.5% Triton X-100 and 20 mM ammonium hydroxide in PBS for 90 min at RT followed by 3 washes with PBS. The efficiency of hydrogel decellularization was confirmed by staining nuclei with DAPI and actin with Phalloidin-TRITC (data not shown).
Cnlm 291 h
Manufactured by Cambridge Isotopes
The CNLM-291-H is a laboratory instrument designed for the analysis of carbon and nitrogen levels in various samples. It features high-precision measurement capabilities and is suitable for use in a range of applications that require accurate determination of carbon and nitrogen content.
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Lab products found in correlation
6 protocols using cnlm 291 h
1
SILAC-Labeled hMSC Hydrogel Decellularization
2
SILAC Labeling Viability Assay for hMSCs
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SILAC αMEM, no nucleosides, no lipoic acid was made up in-house under sterile conditions. Heavy SILAC medium was prepared with 0.1 mg mL−1 heavy isotopically labeled lysine (CNLM-291-H, Cambridge Isotope Laboratories, Inc) and heavy isotopically labeled arginine (CNLM-539-H, Cambridge Isotope Laboratories, Inc). Unlabeled Light SILAC media was prepared with 0.1 mg mL−1l -arginine (A8094, Sigma) and l -lysine (L8662, Sigma). FBS was extensively dialyzed with PBS using Amicon 3K MWCO units (Millipore, UK). Viability of hMSC seeded on tissue culture plastic (TCP) or encapsulated within hydrogels was evaluated using SILAC and control culture media as described in the Supplementary Methods (Supplementary Fig. 20 ).
3
SILAC-Based Quantitative Proteomics
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For quantitative mass spectrometry (MS) analysis, SILAC cells were labeled by culturing them in DMEM without Arg/Lys (A14431-01), 200 µM L-glutamine, 100 U/ml penicillin streptomycin, 110 mg/l (1 mM) sodium pyruvate, and 10% 10 kD dialyzed serum (F0392; Sigma-Aldrich). The media for light labeling was supplemented with Arg 0 (84 mg/ml, A6969; Sigma-Aldrich) and Lys 0 (146 mg/ml, L8662; Sigma-Aldrich), and the media for heavy labeling was supplemented with Arg10 (CNLM-539-H; Cambridge Isotope Laboratories) and Lys8 (CNLM-291-H; Cambridge Isotope Laboratories). Incorporation of the isotopes was confirmed after six passages. siControl and siMASTL#6 silenced samples were prepared two times with light- and heavy-labeled cells, obtaining four experimental replicates. For each independent experiment, siControl and siMASTL-silenced samples were mixed using a label-swap replication strategy (i.e., siControl-light mixed with siMASTL silenced-heavy generates forward replicate 1; siMASTL silenced-light with siControl-heavy generates reverse replicate 1, etc.). 48 h after silencing the cells were lysed with 4% SDS, 100 mM DTT, and 100 mM Tris-HCl, pH 7.6, and boiled for 5 min at 95°C. Samples were sonicated, centrifuged at 13,000 g for 10 min at 4°C, and transferred into a clean tube.
4
SILAC Labeling of hMSC-Laden Hydrogels
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hMSC-laden hydrogels (n = 3) were prepared as described above, except the cell suspension was washed extensively with “heavy” SILAC medium (SILAC αMEM, no nucleosides, no lipoic acid prepared in-house and supplemented with 0.1 mg/mL heavy isotopically-labeled lysine and arginine, #CNLM-291-H, #CNLM-539-H, Cambridge Isotope Laboratories, Inc.) prior to encapsulation. LD and HD hydrogel cultures were cultured for 72 h in “heavy” SILAC medium with 10% dialyzed FBS (against PBS using Amicon 3K MWCO units, Millipore) and 1% antibiotic-antimycotic solution. Samples were decellularized with 0.5% Triton X-100 and 20 mM ammonium hydroxide in PBS for 90 min at RT followed by 3 washes with PBS. The efficiency of hydrogel decellularization was confirmed by staining nuclei with DAPI and actin with Phalloidin-TRITC (data not shown).
5
Labeling Nascent Proteins in Cultured Cells
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In brief, 2 h before collection, cells were washed twice with warm PBS to remove interfering medium and cultured for an additional 2 h with DMEM medium containing 84 mg/l l-arginine ( 13 C6 15 N4 (R10); Cambridge Isotope Laboratories, CNLM-539-H) and 146 mg/l l-lysine ( 13 C6 15 N2 (K8), Cambridge Isotope Laboratories, CNLM-291-H) to label nascent proteins. After labelling culture, the cells were washed three times with warm PBS and lysed with 95 °C hot lysis buffer (100 mM EPPS pH 8.2, 2% sodium deoxycholate, 1 mM TCEP, 4 mM 2-chloracetamide, protease inhibitor tablet mini EDTA-free (Roche)). Samples were then incubated for an additional 5min at 95 °C, followed by sonication for 30 s and a further 10-min incubation at 95 °C.
6
Quantitative Proteomics of SMN and Gemin5 Complexes
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Control, shSMN, and shGemin5 cells were grown in DMEM containing 10% dialyzed FCS and light amino acids L-8662 ([12C]lysine; Sigma-Aldrich) and A-6969 ([12C]arginine; Sigma-Aldrich). After efficient knockdown using doxycycline induction, the control cells were grown in DMEM containing heavy isotope amino acids CNLM-291-H ([13C], [15N]lysine; Cambridge Isotope Laboratories) and CNLM-539-H ([13C], [15N]arginine; Cambridge Isotope Laboratories), whereas the SMN or Gemin5 knockdown cells were grown in medium heavy amino acids DLM-2640 ([2H]lysine; Cambridge Isotope Laboratories) and CLM-2265-H ([13C]arginine; Cambridge Isotope Laboratories) for 24 h. Next, cells were lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2, and protease inhibitor cocktail). After incubation on ice for 10 min, the cells were lysed using 26G needle followed by mild water bath sonication to ensure complete nuclear lysis of the cells and centrifugation at 13,200 rpm for 20 min to remove cell debris. Heavy and medium pulse-labeled cell lysates were mixed in a 1:1 ratio, based on whole protein content estimated by Bradford assay (500-0006; Bio-Rad Laboratories) before processing for MS. siRNA-mediated control and pICln knockdown cells were processed similarly after the transfections.
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