The mycelia of the stable mutants, strain #1, strain #2 and strain #3 were filtered with three-layer gauze, washed with pure water three times, frozen in a cryogenic refrigerator (Forma 900 series, Thermo, Waltham, MA, USA) at −80 °C for 12 h, dried in a vacuum freeze dryer (Labconco, Kansas, MO, USA) for 28 h, ground into LMP in a mortar, and then filtered through a 90 mesh sieve. The mortar and sieve were washed with 95% ethanol three times prior to the next LMP being treated. According to a previously described method [23 (link)], water extracts (10 mL) of treated LMP (0.1 g) of the stable mutants, strain #1, strain #2 and strain #3 were prepared to determine the cordycepin concentration in the cells using HPLC. The residual treated LMP was stored at −20 °C before being used in subsequent experiments.
Vacuum freeze dryer
Manufactured by Labconco
Sourced in United States
The Vacuum Freeze Dryer is a laboratory equipment designed for the process of lyophilization. It creates a low-pressure, low-temperature environment to remove water from samples through the process of sublimation, preserving the structure and composition of the sample.
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Lab products found in correlation
Forma 900 series, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
E coli top10, by Sangon (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
96 well plate, by Greiner (1 mentions)
Inspect f50, by Thermo Fisher Scientific (1 mentions)
Su8010, by Hitachi (1 mentions)
Poremaster gt 60, by Anton Paar (1 mentions)
Qp2010 ultra mass spectrometer, by Shimadzu (1 mentions)
Rtx wax column, by Shimadzu (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
17 protocols using vacuum freeze dryer
1
Quantifying Cordycepin in Fungal Mutants
2
Adherence of VBNC Bacteria to Collagen and Dentine
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The ability of cells to adhere to collagen or dentine was tested by adding bacteria in the VBNC state to dishes containing sterilized collagen fibers or pieces of dentine. The dishes were incubated in an anaerobic culture chamber at 37°C for 48 h. Cells in the exponential growth phase were also cultured as a positive control. We used shaker (180 rpm) during the incubation period to remove the influence of dead cells mixed with the VBNC cells. After incubation, the dishes were washed twice with sterile distilled water to remove nonadherent bacteria. The collagen fibers and dentine pieces were fixed with 2% (v/v) glutaraldehyde for 2 h and washed twice with sterile distilled water. Next, the collagen fibers and dentine pieces were snap-frozen at -80°C for 15 min. The samples were dried in a vacuum freeze dryer (Labconco, Kansas City, MO, USA), gold-coated, and examined by scanning electron microscopy (S-3000N; Hitachi, Tokyo, Japan).
3
Crab Species Composition and Preparation
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Five living female farmed crab species (Figure S5 ), including P. camtschaticus (Paralithodes camtschaticus), E. sinensis (Eriocheir sinensis), C. magister (Cancer magister), P. trituberculatus (Portunus trituberculatus) and C. pagurus (Cancer pagurus), were purchased from a local seafood market in Wuhan, China, in October 2020, with the help of professionals (12 fresh crabs of each species, individually weighed, P. camtschaticus: 1500–1700 g; E. sinensis: 150–200 g; C. magister: 750–800 g; P. trituberculatus: 250–300 g; and C. pagurus: 900–1000 g). The ages of each species of crab shell were similar, and all crab limbs were free of damage. According to a previous study [48 (link)], all crabs were electrocuted through 110 volts and 2 amps of current for 10 s with a Crustastun machine (Studham Technologies, Scotland, UK) and killed immediately. The edible viscera and muscle were manually separated from the crabs, immediately preserved in liquid nitrogen and then lyophilized using a vacuum freeze-dryer (Labconco Corporation, Kansas, MO, USA) with a vacuum of 0.06 MPa. The freeze-dried crab tissues were stored at −80 °C until analysis.
4
Comprehensive Assessment of Coastal Sediment Contamination
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In order to comprehensively understand the contamination status of CPs in the East China Sea and the Yellow Sea, surface marine sediment samples (0–5 cm) were collected in September 2019. A total of 29 sampling locations were selected as shown in Figure 1 , including 5 in the Yellow Sea (S14, S15, S16, S17, and S18) and 24 in the East China Sea (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, and S29). Moreover, 10 (D2, D3, D4, D5, D6, D7, D8, D9, D10, and D11) and 1 (D1) sewage outlets were found in the East China Sea and the Yellow Sea, respectively, and are shown in Figure 1 . The sediment samples were freeze-dried with a vacuum freeze dryer (Labconco Corporation, Kansas City, MO, USA) and then homogenized and stored at −20 °C until extraction.
5
Mycobacterium Tuberculosis Assay Protocol
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CS35 and HRP-goat anti-mouse IgG antibodies were purchased from BEI Resources (USA) and Biodragon (China), respectively. ConA and BSA were obtained from Sigma–Aldrich (USA). Middlebrook 7H9 broth and oleic acid-albumin-dextrose-catalase enrichment (OADC) were purchased from BD Difco (USA). The vacuum freeze dryer was purchased from Labconco (USA). HiPrep 16/60 Sephacryl S-100 HR column was obtained from Cytiva (USA). Moreover, the 96-well plates were purchased from Greiner (Germany). 3,3',5,5'-Tetramethylbenzidine (TMB) substrate was obtained from Millipore (USA) substrate. Alexa Fluor® 594-conjugated ConA was obtained from Invitrogen (USA). All other chemicals were of analytical purity. Mycobacterium tuberculosis H37Rv (ATCC 27294), Mycolicibacterium smegmatis (ATCC 700084), and Mycobacterium bovis BCG (ATCC 35734) were preserved in our laboratory. Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853), and Acinetobacter Baumannii (ATCC 19606) were provided by Jianhui Li (Shanghai Public Health Clinical Center). E. coli Top10 was purchased from Sangon Biotech (China).
6
Velvet Antler Extraction and Analysis
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Velvet antler was obtained from Liaoyuan Dongfeng Sika Deer Farm, (Jilin, China). Fresh VAs were sliced, freeze-dried using a vacuum freeze dryer (-74°C and 12 Pa, LABCONCO, United States), then were comminuted by a grinder. VA powder was mixed with ultrapure water at the mass-volume ratio of 1:10, and ultrasonically extracted for 30 min. Took 50 μl supernatant, and added 450 μl precipitator (methanol: acetonitrile = 1:1). Mixed the two with turbine mixer for 60 s, then centrifuged with 13000 rpm for 10 min. Took 100 μl liquid to do LC-MS analysis.
7
Crab Muscle Extraction and Preparation
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The 48 crabs were collected from the freezer 2 days from the day of arrival and allowed to thaw, and then 6–8g of muscle was removed from the appendage carapaces of each crab. These muscle tissues were dried using a vacuum freeze dryer (Labconco Corp., Kansas City, MO, United States) and grounded into powder form with a mortar and pestle. The dried samples were kept in ziplock plastic bags and stored at −20. Thereafter, they were sent to the lab for FA profile analysis.
8
Lyophilized PRP Gel Microstructure Analysis
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The GO/Alg gel with PRP was mixed and aliquoted into sterile EP tubes, after 1 h of standing at room temperature, the gel sample with a complete appearance and satisfactory strength was placed in the refrigerator at −80°C for 24 h, and then the frozen sample was placed into a vacuum freeze dryer (−88°C, 0.011 kPa, Labconco Co., US) for 24 h to obtain fluffy lyophilized samples. Sharply cut the middle part of the lyophilized sample for vacuum gold spraying, and then observe the microstructure of the gold-plated sample with SEM.
9
Pegylation of Mesoporous Silica Nanoparticles
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The solution of PEG600-2NHS (10 mg) in 1 mL of dichloromethane was added to the suspension of MSNPs (100 mg) in 4 mL of DMSO, and the stirring of the reaction mixture was performed overnight at RT. Then, nanoparticles were collected by centrifugation at 5000 ×g and washed with ethanol (×3). The final product was dried with the Labconco vacuum freeze-dryer (Kansas City, US).
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The solution of PEG600-2NHS (10 mg) in 1 mL of dichloromethane was added to the suspension of MSNPs (100 mg) in 4 mL of DMSO, and the stirring of the reaction mixture was performed overnight at RT. Then, nanoparticles were collected by centrifugation at 5000 ×g and washed with ethanol (×3). The final product was dried with the Labconco vacuum freeze-dryer (Kansas City, US).
10
Drug Loading of Mesoporous Silica Nanoparticles
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To prepare for drug loading, the SUN solution in DMSO (55 mg/2 mL) was added to the suspension of MSNP-PEG in DMSO (55 mg/1 mL) and the final suspension was stirred at RT overnight. NSs were centrifuged at 5000 ×g and separated. The supernatant which contained the unloaded SUN was analyzed to determine loading efficiency. SUN-loaded MSNPs were dried using the Labconco vacuum freeze-dryer (Kansas City, US).
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