After fixation in 4% PFA, samples were incubated in 4% acrylamide (Sigma-Aldrich Inc., St. Louis, MO, United States) in 0.1 M PBS (A4P0) at 4°C for 24 h and then in 0.25% VA-044 polymerization initiator (Wako Chemicals United States, Inc., Richmond, VA, United States) in 0.1 M PBS at 4°C for 24 h. Samples were embedded in a nitrogen gas atmosphere for 5 min, and placed in a shaking incubator at 37°C for 12 h. The mPACT-A samples were transferred to A4P0 at 37°C for 6 h. Samples processed with psPACT were incubated in clearing buffer (8% sodium dodecyl sulfate (SDS; Affymetrix Inc., OH, United States) in 0.1 M PBS, pH 8.0) at 37°C until they became transparent. Samples processed with mPACT were incubated with clearing buffer supplemented with 0.5% α-thioglycerol (Sigma-Aldrich Inc., St. Louis, MO, United States) at 37°C until they became transparent. Upon achieving transparency, all samples were washed with 0.1 M PBS for 2 h and subsequently stored in nRIMS solution [0.8 g/ml Nycodenz (Axis-Shield Density Gradient Media, Oslo, Norway), 0.01% sodium azide (Sigma-Aldrich Inc., St. Louis, MO, United States), and 0.1% Tween-20 (Georgiachem, Suwanee, GA, United States) in 0.1 M PBS, pH 7.5].
α thioglycerol
Manufactured by Merck Group
Sourced in United States, Italy
α-thioglycerol is a chemical compound used in various laboratory applications. It serves as a reducing agent and can be utilized in synthetic organic chemistry reactions. The core function of α-thioglycerol is to provide a source of thiol groups for chemical transformations.
Automatically generated - may contain errors
Lab products found in correlation
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (5 mentions)
Fructose, by Merck Group (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (3 mentions)
L glutamine 200 mm, by Merck Group (3 mentions)
L ascorbic acid, by Merck Group (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
D fructose, by Avantor (2 mentions)
Phire tissue direct pcr master mix, by Thermo Fisher Scientific (2 mentions)
α mem glutamax medium, by Thermo Fisher Scientific (2 mentions)
Insulin transferrin selenium 100, by Thermo Fisher Scientific (2 mentions)
Live dead fixable far red stain, by Thermo Fisher Scientific (2 mentions)
Amphotericin b 250 μg ml, by Merck Group (2 mentions)
Eosin y, by Merck Group (2 mentions)
Type 45 ti rotor, by Beckman Coulter (2 mentions)
Streptomycin, by Merck Group (2 mentions)
40 protocols using α thioglycerol
1
Tissue Clearing and Preparation for Imaging
2
Culturing Mouse and Human Lymphoma Cells
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Eμ-Myc mouse lymphoma cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), 50 μM β-mercaptoethanol (Sigma-Aldrich), 100 μM asparagine (Sigma-Aldrich), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37°C and 10% CO2. OP9 cells were cultured in αMEM (Gibco) supplemented with 20% heat-inactivated FBS, 1 mM glutamine (Gibco), 10 mM Hepes (Gibco), 1 mM sodium pyruvate (Gibco), 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. Human cell lines were cultured in a humidified incubator at 37°C and 5% CO2. Virus-producing 293T cells were maintained in DMEM supplemented with 10% FBS. Approximately 6 h prior to transfection, 293T cells were cultured in DME glutamax (Gibco) supplemented with 10% FBS and 25 mM Hepes. Rael-BL, Ramos-BL, Sav-BL, and BL-31 human Burkitt lymphoma-derived cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 50 μM α-thioglycerol (Sigma-Aldrich), and 20 nM bathocuproine disulfonic acid (BCS) (Sigma-Aldrich). X50-7 and Awia lymphoblastoid cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 1 mM glutamine.
3
Optical Clearing of Immunostained Biopsies
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To prepare D-fructose solutions for optical clearing, 20, 50, and 100% (w/v) solutions of D-fructose were prepared in 10 mM phosphate buffer pH 7.8 to a final volume of 10 ml. 30 μl of α-thioglycerol (Sigma Aldrich) was added to each D-fructose solution. Immunostained core needle biopsies were incubated sequentially in 10 ml of 20, 50, and 100% D-fructose solutions for 30 min, 30 min, and 1 h at 25°C with gentle agitation in 20 ml glass vials, respectively. The immunostained cores and agarose were then mounted between coverslips in 100% (w/v) D-fructose solutions.
4
Fructose-Mediated Tissue Clearing Protocol
5
Culturing Human Mast and Erythroleukemic Cells
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The human mast cell line HMC-1 (Dr. Joseph Butterfield, Mayo Clinic, Rochester, MN, USA) and the erythroleukemic cell line TF-1 (ATCC: CRL-2003) were cultured in a 37°C humidified incubator with 5% CO2. HMC-1 was cultured in Iscove's Modified Dulbecco's Medium (IMDM), while TF-1 was cultured in RPMI-1640. Both media were supplemented with 10% foetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L -glutamine. In addition, the medium for HMC-1 also contained 1.2 mM α-thioglycerol and the medium for TF-1 was supplemented with 5 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (all reagents were from Sigma-Aldrich, St Louis, MO, USA).
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
6
Aqueous Immersion Clearing Solution
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For simple immersion clearing, AICI (Aqueous Immersion Solution for tissue Clearing and Imaging) reagent was prepared as 20% (wt/vol) N-methyl-D-glucamine (#M2004, Sigma-Aldrich, St. Louis, MO, USA), 30% (wt/vol) TDE (#166782, Sigma-Aldrich, St. Louis, MO, USA), 45% (wt/vol) Iodixanol (#D1556, Sigma-Aldrich, St. Louis, MO, USA), 0.2% (wt/vol) Triton X-100 (20%) (#T8787, Sigma-Aldrich, St. Louis, MO, USA), 0.5% (wt/vol) α-thioglycerol (#88640, Sigma-Aldrich, St. Louis, MO, USA), 1% (wt/vol) Triethanolamine (#90278, Sigma-Aldrich, St. Louis, MO, USA) dissolved in pure DW (#10977-015, Invitrogen, Carlsbad, CA, USA). After mixing with a magnetic stirrer for 2 h at 25 °C, the solution was filtered using a bottle-top vacuum system into an amber bottle. The reagent was stored for several months at 25 °C.
7
Optical Clearing of Immunostained Biopsies
8
Establishing Stable HeLa Cell Lines
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HeLa cells were transfected as indicated in Supplementary Table S24 . At 3 days post-transfection, the cells were transferred into wells of six-well plates (Greiner Bio-One) and were subsequently exposed to 1 μg ml−1 puromycin (Invitrogen, Cat. No.: A11138-03) for 7 days. The resulting puromycin-resistant HeLa clones were identified through colony-formation assays using standard Giemsa or Crystal violet staining protocols. In addition, parallel cultures of puromycin-resistant HeLa cell populations were seeded at a density of 0.3 cells per well in wells of 96-well plates (Greiner Bio-One). The resulting single cell-derived clones were then sub-cultured for ∼3 weeks in DMEM supplemented with 5% FBS, 1 μg ml−1 puromycin, 50 nM α-thioglycerol (Sigma-Aldrich; Cat. No.: M6145) and 0.02 nM bathocuproinedisulfonic acid disodium salt (Sigma-Aldrich; Cat. No.: B1125). Subsequently, genomic DNA of randomly collected single cell-derived clones was extracted and analysed by junction PCR using Phire™ Tissue Direct PCR Master Mix (Thermo Fisher Scientific, Cat. No.: F-107L) according to the manufacturer's protocols. The PCR primer pairs, composition of the PCR mixtures and cycling parameters are specified in Supplementary Tables S35 and S36 , respectively.
9
Cell culture protocol for viability
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Conventional culture dishes that are 50 mm in diameter were purchased from Falcon (Sigma-Aldrich, Milan, Italy). Leibovitz’s L-15 medium, α-MEM GlutaMAX medium, insulin transferrin selenium (ITS) 100×, and LIVE/DEAD Fixable Far Red stain were purchased from Invitrogen (Milan, Italy). Penicillin-streptomycin 100×, amphotericin B 250 μg/mL, bovine serum albumin, L-ascorbic acid, L-glutamine 200 mM, Hoechst 33342, fructose, and α-thioglycerol were purchased from Sigma-Aldrich (Milan, Italy). Mayers’s hematoxylin and paraffin wax were purchased from Carlo Erba (Milan, Italy).
10
Mast Cell Culture and Characterization
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HMC-1.1 were used for experiments on mast cells [83 (link)]. An aliquot of HMC-1.1 cells had previously been generously gifted by Dr Butterfield to PTKS allowing us to conduct in vitro mast cell investigations. Cells were cultured in IMDM medium (Gibco, Paisley, UK) supplemented with 10% calf serum (Hyclone, Logan, UT, USA), using 162 cm3 cell culture flasks (Corning Incorporated, Costar®, Corning, NY, USA) and incubated at 37 °C with 5% of CO2. Cells were split and collected in 6 wells culture plates (Corning Incorporated, Costar®, Corning, NY, USA) at a density of 1 × 106 cells/mL. Each well contained 3 mL of cell suspension. The HMC-1.1 cells were maintained in IMDM medium, supplemented with 10% calf serum, plus α thioglycerol (Sigma, Gillingham, UK). Cell counting was performed using Countess II Life Technologies Cell Counter (Countess™ Invitrogen, Waltham, MA, USA), by Trypan blue staining.
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