C3H10T1/2, MC3T3-E1, and 3T3-L1 cells were purchased from American Type Culture Collection. C3H10T1/2 and 3T3-L1 cells were maintained in DMEM (GIBCO-BRL, CA, USA), whereas MC3T3-E1 cells were maintained in MEMα (catalog no. A1049001, GIBCO-BRL, CA, USA). Culture media were supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. C3H10T1/2 cells were transfected with pcDNA3.1-ZFP36L1 and selected with G418 (1 mg/ml). Twenty two antibiotic-resistant clones were harvested and examined for ZFP36L1 overexpression. Clones 2 and 21 which expressed high level of ZFP36L1 were selected for experiments. The femurs and bone marrow mesenchymal stem cells isolated from the femurs of adult and aged Fisher 344 rats were kind gifts from Dr. Chun-Chin Liang. For the isolation of RNA from rat femurs, the femurs were cryogenically pulverized at -195 °C by liquid N2 using a SPEX 6770 Freezer/Mill (SPEX SamplePrep, NJ, USA). Total RNA was then extracted using Trizol Reagent (Life Technology, MD, USA).
6770 freezer mill
Manufactured by SPEX
Sourced in United States, United Kingdom
The 6770 Freezer/Mill is a laboratory instrument designed for cryogenic grinding and milling of a wide range of sample materials. It uses liquid nitrogen to rapidly cool and freeze samples, allowing for efficient size reduction and homogenization.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Freezone triad, by Labconco (2 mentions)
Polycaprolactone, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Truseq library preparation kit v3, by Illumina (2 mentions)
C3h10t1 2, by Thermo Fisher Scientific (1 mentions)
Mc3t3 e1, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mc3t3 e1, by American Type Culture Collection (1 mentions)
C3h10t1 2, by American Type Culture Collection (1 mentions)
Emulsiflex c3, by Avestin (1 mentions)
Dd2 spectrometer, by Agilent Technologies (1 mentions)
Quanti fast one step rt pcr kit, by Qiagen (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Mirvana rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Spotlight 400 spectrometer, by PerkinElmer (1 mentions)
Ribopure rna purification kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent solution, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
28 protocols using 6770 freezer mill
1
Cell Line Maintenance and Transfection
2
Lyophilized ECM Particle Preparation
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Lyophilized ECM samples were cryogenically pulverized at −195°C under liquid N2 using a SPEX 6770 Freezer/Mill (SPEX SamplePrep, Metuchen, NJ) according to the manufacturer's instructions. The resulting powder was resuspended in anhydrous ethanol (ETOH) and homogenized at 170 MPa using an EmulsiFlex C3 with an in-line heat exchanger (Avestin, Inc., Ottawa, ON, Canada). The suspension was filtered to exclude particles greater than 1 μm in diameter. Filtered ECM particles were introduced into anhydrous dimethyl sulfoxide (DMSO) and the ethanol was removed at room temperature using a high vacuum.
3
Metabolite Extraction and NMR Analysis
4
Cartilage RNA Extraction and qRT-PCR
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Articular hip and knee cartilage was snap‐frozen in liquid nitrogen and pulverized using a 6770 Freezer/mill (Spex Sample Prep). Total RNA was extracted from both powdered cartilage and primary chondrocytes using TRIzol and further purified using RNeasy columns. RIN values were >7, and 260:280 ratios were >1.7. Custom primers and FAM‐labeled probes were designed using Primer Express 3 software (Life Technologies) for qRT‐PCR. The qRT‐PCR was performed from 25 ng of total RNA in a one‐step reaction (QuantiFast One‐Step RT‐PCR kit; Qiagen) using a Roche LightCycler 480 II. The relative expression of lncRNAs was determined using the ΔΔCt method, following normalization to 18S RNA. GAPDH expression (relative to 18S RNA) was comparable between non‐OA and OA cartilage in both hip and knee samples (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.39520/abstract ).
5
Fish Biometrics and Tissue Preparation
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Upon return to the laboratory, standard length (anterior tip of premaxilla to posterior border of hyperal plate) of each fish was measured with digital calipers (±0.01 mm). Stomach contents were removed with stainless steel forceps and discarded, and the stomach was placed back into the cavity of the fish. Following removal of stomach contents, whole body mass was obtained ±0.001 g. Sex was determined based on inspection of gonads.
Fish were oven dried at 50°C for approximately 48 hours. Dried samples were homogenized by cryogenic (liquid nitrogen immersion) grinding using a cryogenic impact mill (6770 Freezer Mill; Spex, Metuchen, NJ, USA) and placed in sterile 25 mL vials. Homogenized samples were placed in the drying oven at 50°C for approximately 48 hours to remove any residual moisture obtained during cryogenic grinding.
Fish were oven dried at 50°C for approximately 48 hours. Dried samples were homogenized by cryogenic (liquid nitrogen immersion) grinding using a cryogenic impact mill (6770 Freezer Mill; Spex, Metuchen, NJ, USA) and placed in sterile 25 mL vials. Homogenized samples were placed in the drying oven at 50°C for approximately 48 hours to remove any residual moisture obtained during cryogenic grinding.
6
Tissue Biopsy RNA Extraction Protocol
7
Preparation and FTIR Analysis of Collagen-Elastin Pellets
8
Genomic DNA Extraction from Powdered Teeth
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Samples were pulverized in liquid nitrogen with a 6770 Freezer/Mill (SPEX SamplePrep, LLC, Metuchen, USA). Powder teeth (0.5 g) were demineralized and lysed in a buffer containing 500 µL of 0.5 M EDTA, 35 µL SDS (10%) and 100 µL of proteinase K (20 mg/mL). Following 24-h incubation at 37 °C with continuous agitation (300 rpm), the tubes were centrifuged at 13,000 rpm for 3 min. The supernatants were transferred and mixed with 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged again at 13,000 rpm for 3 min. Then, the supernatants were taken and added to Centricon-100 concentrators (Centricon-100, Millipore, Bedford, MA) and centrifuged at 2500 rpm for 20 min. Finally, concentrators were placed into 1.5 mL microcentrifuge tubes and DNA was recovered in 30 µL of the elution buffer (10 mM Tris/HCl, pH 8.5) after centrifugation at 2500 rpm for 20 min. Blanks were included in DNA extraction procedures and PCR amplifications. Samples were stored at − 20 °C before DNA analysis.
9
Cryogenic Milling of Murine Femurs
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Both femur diaphysis per mouse were pooled and pulverized in liquid nitrogen using a 6770 Freezer Mill (SPEX CertiPrepFreezerMill, Stanmore, London, UK). The cryogenic milling was carried out under mild conditions (cycles: 2; run time: 2 min; rate: 9 cps) to avoid altering the crystallinity of the materials or the spectral levels of the compounds under study. The resulting powder (50–100 µg particle size) was collected (~250 mg) and kept in a −80° freezer until XRD and ATR-FTIR analyses.
10
Cryogenic Pulverization of PLGA Nanofibers
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Electrospun nanofibre mesh of PLGA was subjected through the cryogrinding process as previously described64 (link) with some modification to the obtained ground powder of nanofibres. A cryogenic impact grinder (6770 Freezer Mill, Spex, USA) with a self-contained liquid nitrogen bath (4–5 L) was used. About 0.2 g of PLGA nanofibre mesh was cut into the small pieces of unit cm2 size and put into a 25 ml polycarbonate grinding vials for pulverization. After a pre-cooling period (5 min), six working cycles were used for each grinding. Each cycle consisted of grinding and re-cooling periods (1 min). The applied impact bar frequency was 14 Hz. The ground fibre was dispersed in ethanol and filtered through sterile 70 µm sieve for the smaller and uniform size of CPN. Dry powder of CPN was collected after complete evaporation of ethanol under the hood.
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