For isolation of anagen cells, skins were first incubated in 0.25% collagenase (Sigma) in HBSS (GIBCO) at 37°C for 1–2 hr to digest the dermis. Next, the digested tissues were centrifuged and resuspended in 0.25% Trypsin (GIBCO) and incubated at 37°C for 20 min with gentle shaking. Single-cell suspensions were obtained by pipetting gently and filtering through 70 μm strainers, followed by 40 μm strainers. Staining buffer (PBS with 5% FBS treated with Bio-Rad Chelex to remove calcium) was added to inactivate Trypsin, and cells were collected by centrifugation for 5 min at 300 × g. Cell suspensions were incubated with the appropriate antibodies diluted in staining buffer for 15 min at 4°C. For the isolation of telogen cells, subcutaneous fat was removed from skins with a scalpel. Skins were placed on 0.25% Trypsin at 37°C for 30 min with the dermis side facing down. Skins were then gently scalped from the epidermis to isolate epidermal cells and filtered, centrifuged, and stained as above. The following antibodies were used: CD34–Alexa 660 (eBioscience), α6–phycoerythrin (eBioscience), Sca1–PE-Cy7 (eBioscience), and c-Kit-APC (eBioscience).
Trypsin
Manufactured by Bio-Rad
Sourced in United States
Trypsin is a proteolytic enzyme that is commonly used in cell culture applications to detach adherent cells from the growth surface. It functions by cleaving peptide bonds at the carboxyl side of lysine and arginine residues, thereby disrupting the cell-substrate interactions and allowing cells to be suspended for further processing.
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Lab products found in correlation
Formic acid, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Bio-Rad (2 mentions)
Sca 1 pe cy7, by Thermo Fisher Scientific (1 mentions)
C kit apc, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Mayer hematoxylin, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Clusterin, by R&D Systems (1 mentions)
Gelsolin, by Abcam (1 mentions)
Casein, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Nh4oh, by Merck Group (1 mentions)
Iodoacetamide, by Bio-Rad (1 mentions)
Trypsin, by Promega (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Milli q water, by Merck Group (1 mentions)
8 protocols using trypsin
1
Isolation of Anagen and Telogen Hair Follicle Cells
2
Proteomic Analysis of Cell Lines
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The following reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA): Coomassie Brilliant Blue R-250, dithiothreitol (DTT), urea, trypsin, glycerol, glacial acetic acid, alpha-cyano-4-hydroxycinnamic acid, acetonitrile, sodium carbonate, HAuCl4, casein, and Ponceau S. We have purchased ReadyStrip (IPG strip pH 4–7) from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Primary mouse monoclonal antibody or rabbit polyclonal antibodies against gelsolin, calpain, peroxire-doxin-2 (Abcam, Cambridge, UK), PEBP, LDH-B, crystalline (Abgent, San Diego, CA, USA), anexxin-2 and LXRα and CRY7B1 (Novus, Littleton, CA, USA), and clusterin (R&D systems, Inc., Minneapolis, MN, USA) were used. We have purchased Mayer hematoxylin from Richard-Allan Scientific (Thermo Fisher Scientific, Waltham, MA, USA) and immunohistochemistry (IHC) reagents from Biocare Medical (Concord, CA, USA). All cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).
3
Proteomic Profiling of Extracellular Vesicles
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EVs A, EVs N or EVs N+A, equivalent to 100 µg of protein each, were run on 12% polyacrylamide gels and run just enough time to allow the mixture of proteins to enter the stacking gel and concentrate as a coarse band. After staining with Bio-Safe Coomasie G-250 stain (BioRad), the bands were cut under sterile conditions and subjected to in-gel trypsin digestion. Briefly, gel pieces were washed with 50 mM ammonium bicarbonate (Acros) in 50% acetonitrile (Fisher), reduced with dithiothreitol (Acros) and alkylated with iodoacetamide (Sigma), washed again, and impregnated with ~75 µL of 6 ng/µL trypsin (trypsin gold; Promega) solution overnight at 37°C. The resulting peptides were extracted using solutions of 50% and 80% acetonitrile (ACN) with 0.5% formic acid (Millipore), and the recovered solution was dried down in a vacuum concentrator.
Dried peptides were dissolved in 60 µL of 0.1% trifluoroacetic acid (TFA, Sigma), and desalted using 2-core MCX stage tips (3M, 2241) (Rappsilber et al., 2003 (link)). The stage tips were activated with ACN followed by 3% ACN with 0.1% TFA. Next, samples were applied, followed by two washes with 3% ACN with 0.1% TFA, and one wash with 65% ACN with 0.1% TFA. Peptides were eluted with 75 µL of 65% ACN with 5% NH4OH (Sigma), and dried.
Dried peptides were dissolved in 60 µL of 0.1% trifluoroacetic acid (TFA, Sigma), and desalted using 2-core MCX stage tips (3M, 2241) (Rappsilber et al., 2003 (link)). The stage tips were activated with ACN followed by 3% ACN with 0.1% TFA. Next, samples were applied, followed by two washes with 3% ACN with 0.1% TFA, and one wash with 65% ACN with 0.1% TFA. Peptides were eluted with 75 µL of 65% ACN with 5% NH4OH (Sigma), and dried.
4
Proteomics Analysis of Pnpla5 Knockout Rats
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For the proteomics quantitative analysis, two groups of 3-month-old rats (n = 3), Pnpla5+/+ andPnpla5−/− were analyzed. The rats were euthanized by cervical dislocation under deep anesthesia, and the testes were recovered and stored in liquid nitrogen. Total protein was isolated as previously described [19 (link)]. Protein concentrations were measured by the Bradford assay using bovine serum albumin (BSA) as the standard. For trypsin digestion and iTRAQ labeling, 200 µg of protein from each sample was reduced with 25 mM DTT (Bio-Rad, California, USA) at 37 °C for 1 h, alkylated with 50 mM iodoacetamide (Bio-Rad, California, USA) for 30 min at room temperature in the dark, and then digested with 4 µg trypsin (Promega, Wisconsin, USA) at 37 °C overnight. The digested samples were labeled with iTRAQ reagents according to the manufacturer’s instructions. In brief, labels 114–116 and labels 117–119 were used for the relative abundance of three Pnpla5−/− and Pnpla5+/+testicular tissues, respectively. The labeled samples were then mixed and vacuum-dried.
5
Trypsin-Mediated Protein Extraction
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Protein spots of interest were excised from the gel using a sterile blade and washed three times with MilliQ water (Merck Millipore). The gel spots were destained twice with 0.2 ml 100 mM NH4HCO3 in 50% acetonitrile (ACN) for 45 min at 37°C prior to being dehydrated in 100% ACN for 5 min. The spots were then incubated with 10 µl of 10 µg/ml trypsin (Bio-Rad Laboratories, Inc.) at room temperature for 1 h, followed by incubation at 37°C overnight in 20 µl digestion buffer (40 mM NH4HCO3 in 10% ACN; Keygen Biotech). The liquid was then removed. The tryptic peptides were extracted twice using 50 µl of 50% ACN with 5% trifluoroacetic acid (TFA; Keygen Biotech) by sonication (JY98-IIIN; Ningbo Scientz Biotechnology Co., Ltd, Zhejiang, China) for 15 min. All extracts were then pooled and dried in a Speed Vac (Keygen Biotech) at room temperature. The peptides were desalted using C18 Zip Tips (EMD Millipore, Billerica, MA, USA) and reconstituted in 5 µl 70% ACN with 0.1% TFA.
6
MTT Assay for Cell Viability
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque Inc (Kyoto, Japan). Cisplatin, Lansoprazole, sodium deoxycholate (SDC), chloroacetamide (CAA), formic acid and ethyl acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). DTT and trypsin were from BioRad (Hercules, CA, USA) and Promega (Madison, WI, Wisconsin) respectively. The cell culture media, DMEM nutrient mixture F-12 (DMEM-F12), were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies used for western blotting include CD63, CD9, CD81, Na+/K+ ATP1B3, EGFR were from Santa Cruz Biotechnology Inc (Dallas, TX, USA), while antibodies against HSC70 and GAPDH were from Cell Signaling Technology (Denver, MA, USA) and Beta-actin from Sigma Aldrich (St. Louis, MO, USA).
7
Quantifying RIG-I Activation in A549 Cells
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To monitor RIG-I activation, A549 cells were transfected with 200 ng/ml of tRNA (negative control), 5'-triphosphorylated double-stranded RNA (positive control; InvivoGen, Toulouse, France) or the endogenous RNase fragment (eRL) using Endofectin (Biocat, Heidelberg, Germany). After 2 h, cells were subjected to RIG-I activation assay as previously described (26 ). Briefly, one part of the lysate was left untreated (input control) and the other part was subjected to limited protease digestion. Therefore, the sample was digested for 10 min with 0.2 μg/μl TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C. The digestion was stopped by adding 4-fold sample buffer (200 mM Tris–HCl pH 6.8, 8% SDS, 40% glycerol, 25% β-mercaptoethanol, 0.4% Bromphenol Blue) and boiling for 5 min at 100°C. Samples were subjected to 15% SDS-PAGE and western blot analysis using mouse monoclonal anti-RIG-I antibody (clone ALME-1, Adipogen, San Diego, California). Band intensity was quantified using Image Lab 5.2.1 (BioRad, Hercules, California) and activated RIG-I represents the ratio of trypsin-resistant RIG-I to undigested RIG-I. Means of three independent experiments are shown. P values were determined using a paired two-tailed Student's t-test.
8
2-DE and MS/MS Protein Identification
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2-DE and MS/MS analysis was performed as described previously [54] (link). Briefly, cells were dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.2% pH3–10 ampholyte, Bio-Rad, USA) in presence of protease inhibitor (Sigma). Samples were loaded into IPG strips (17 cm, pH3–10NL, Bio-Rad) using a passive rehydration method, and then subjected to isoelectric focusing (Bio-Rad). The second dimension separation was performed using 12% SDS-PAGE after equilibration. The gels were stained with CBB R-250 (Bio-Rad). Identification and quantitation of protein spots in the gel was achieved using PDQuest software (Bio-Rad).
In-gel protein digestion was performed using mass spectrometry grade trypsin according to the manufacturer’s instructions. The gel spots were destained with 100 mM NH4HCO3/50% acetonitrile (ACN) and dehydrated with 100% ACN. The gels were then incubated with trypsin (Promega, V5280), followed by double extraction with 50% ACN/5% trifluoroacetic acid (TFA). The peptide extracts were dried in a speed-VAC concentrator (Thermo), and subjected to mass spectrometric analysis using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source.
In-gel protein digestion was performed using mass spectrometry grade trypsin according to the manufacturer’s instructions. The gel spots were destained with 100 mM NH4HCO3/50% acetonitrile (ACN) and dehydrated with 100% ACN. The gels were then incubated with trypsin (Promega, V5280), followed by double extraction with 50% ACN/5% trifluoroacetic acid (TFA). The peptide extracts were dried in a speed-VAC concentrator (Thermo), and subjected to mass spectrometric analysis using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source.
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