For retroviral production, 293T cells at 80% confluence were trypsinized and single cell suspension was prepared in PBS for transfection by electroporation as described [8 (link)]. Briefly, a 400-μl mixture containing 107 cells and 15 μg of plasmid DNA was electroporated with square wave delivered at 110 V and 25 ms by the Easyject electroporator (Bio-Rad). The15 μg of plasmid DNA combined 4 plasmids (1.7 μg of pAdvantage, 2.6 μg of pVSV-G, 4 μg of pGag-pol, and 6.7 μg of pLegfp, pLcherry or pGTgfp, respectively). Electroporated cells were incubated on ice for 15 min, centrifuged for 5 min at 2000 rpm (Centrifuge 5415C, Eppendorf), resuspended in DMEM-10% FCS and seeded onto 10-cm dishes for culture. The culture medium containing RVs was collected at 48h after transfection, filtrated through a 0.45-μm filter (Sartorius) and concentrated by centrifugation at 4°C for 150 min at 20,000 rpm (Beckman J2-21). The RV pellet was gently resuspended in 100-μl of ESM4 medium for storage at -80°C and cell infection.
Centrifuge 5415c
Manufactured by Eppendorf
Sourced in Germany
The Centrifuge 5415C is a compact benchtop centrifuge designed for routine applications in the laboratory. It features a fixed-angle rotor that can accommodate 18 microcentrifuge tubes with a maximum capacity of 2 mL each. The centrifuge operates at a maximum speed of 14,000 rpm, providing a relative centrifugal force (RCF) of up to 16,873 x g. The unit is equipped with an electronic control system and a time setting of up to 99 minutes.
Automatically generated - may contain errors
Lab products found in correlation
Haake rheostress 1, by Thermo Fisher Scientific (4 mentions)
Malvern nano zs zetasizer, by Malvern Panalytical (4 mentions)
Superose 12 gl column, by GE Healthcare (4 mentions)
Kta purifier 900 series, by GE Healthcare (3 mentions)
Sulphanilic acid, by Merck Group (2 mentions)
Aspergillus species nad p h, by Merck Group (2 mentions)
N 1 naphthyl 1 ethylendiaminedihydrochloride, by Merck Group (2 mentions)
Trichloroacetic acid, by Merck Group (2 mentions)
Phosphoric acid, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
J2 21, by Beckman Coulter (1 mentions)
0.45 μm filter, by Sartorius (1 mentions)
K2edta, by BD (1 mentions)
Thermomixer 5436, by Eppendorf (1 mentions)
Fmax microplate spectrofluorometer, by Molecular Devices (1 mentions)
Opus 8, by Bruker (1 mentions)
Bioatr 2 with znse atr element, by Bruker (1 mentions)
Tensor 27 spectrometer, by Bruker (1 mentions)
Lxsahm, by R&D Systems (1 mentions)
25 protocols using centrifuge 5415c
1
Retroviral Production by Electroporation
2
Dynamic Light Scattering and Rheology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 18
Dynamic light scattering (DLS) was performed using a Malvern Nano Zetasizer ZS (Malvern Instruments Ltd Enigma Business Park, Grovewood Road, Malvern, Worcestershire, UK. WR14 1XZ) and a Haake Rheostress 1 (Thermo Fisher Scientific, Karlsruhe, Germany) equipped with a cone with 60 mm diameter/0.5° angle for buffer viscosity measurements.
All samples were centrifuged (Centrifuge 5415C, Eppendorf, Vienna, Austria) for 5 min at 10.000 rpm to determine the hydrodynamic diameter of a protein. 60 μL of sample were filled into a ZEN0040 disposable micro cuvette and viscosity of buffer was determined by Rheostress 1. This parameter is used for analyzing effective size of proteins by DLS. Operation temperature was 25° C. with an equilibration time of 2 minutes. The proteins angle was set to 173° backscatter to measure the size of and 3 runs per sample were performed to average the results.
Samples were measured by increasing temperature mode to monitor the influence of temperature on a protein. Measurement procedure was similar to a normal size measurement, except for different temperatures with an increasing value of 1° C./min from 15° C. to 80° C. and an equilibration time of 2 min. A DTS2145 low volume glass cuvette was used for these temperature ramps.
3
Soluble Sugar Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the extraction of the neutral fraction of water-soluble compounds (referred to as soluble sugar hereafter), 51 ± 1 mg of each sample were transferred to 2 mL Eppendorf vials. The extraction was based on Wanek et al. (2001) (link). Each sample was vortexed after receiving 1.5 mL of MilliQ water. All samples were placed in a shaking water bath (80°C) for a total of 30 minutes and were vortexed after 15 minutes. Vials were then centrifuged at 12000 g (Centrifuge 5415 C, Eppendorf) for two minutes. The pellet was used for starch extraction as described below. One milliliter of the supernatant was removed and pipetted on top of a column filled with anion (DOWEX 1X8, 100-200 mesh) and cation exchange (DOWEX 50WX8, 100-200 mesh) resin and eluted with 25 mL MilliQ water, to obtain the soluble sugar. These solutions were then freeze dried and resuspended in 1 mL of MilliQ water and stored at -20°C until further analysis.
4
Longitudinal Metabolic Monitoring in Pregnancy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Body weights were recorded weekly during the study, beginning at eight weeks of age and in early (GD4-7), mid (GD14-17) and late pregnancy (GD19-20). Morning blood samples were also collected, mixed with anti-coagulant (K2 EDTA, BD, Franklin Lakes, NJ, USA) and remained on ice until centrifugation (Eppendorf Centrifuge 5415C, Germany, 16,000× g, 5 min). Plasma was stored at −20 °C until being analyzed for glucose (Trinder assay kit, Genzyme Diagnostics, Charlottetown, PEI, Canada), insulin (Rat Ultrasensitive ELISA Immunoassay kit, ALPCO Diagnostics, Salem, NH, USA), and triglycerides (Triglyceride-SL assay kit, Genzyme Diagnostics, Cambridge, MA, USA).
5
Plasma Amino Acid Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were prepared by mixing 400 μL plasma and 400 μL sample dilution solution (Lithium Loading Buffer Kit, Biochrom Ltd., Cambridge, UK) which included the internal standard Norleucine 200 nmol/mL) in an Eppendorf tube. To this, 200 μL 10% (w/v) 5-sulfosalicylic acid (SSA) solution for the deproteinization were added and the sample was deposited in the refrigerator for 20 min at 4 °C. Afterwards, the sample was centrifuged (Eppendorf centrifuge 5415 C) at 11,000× g for 5 min. The supernatant was centrifuged a second time with a nylon membrane filter, pore size 0.22 µm (Laborservice Onken, Gruendau, Germany) at 11,000× g for 2 min.
6
Nitrite and Nitrate Detection by Griess Reaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A modified Griess reaction was used to detect nitrite and nitrate [36 (link),37 (link)]. The NO levels in samples were indirectly measured after first converting nitrates to nitrites with a nitrate reductase treatment (Aspergillus species NAD [P] H, Sigma, UK) and NADPH β-nicotinamide adenine dinucleotide phosphate (Sigma Diagnostics, St. Louis, USA). Griess reagent [5% phosphoric acid, 1% sulphanilic acid and 0.1% N-(1-naphthyl-1)-ethylendiaminedihydrochloride, all from Sigma, UK, dissolved in 100 mL deionized water] was added and proteins were subsequently precipitated by trichloroacetic acid (BDH, England). The tube contents were mixed and centrifuged (Eppendorf centrifuge 5415 C, Germany); two samples of each supernatant were transferred to a flat-bottomed microplate and their absorbances were read at 520 nm using a microplate reader (SpectraMax, Molecular Devices Inc). NO values were calculated from standard calibration plots.
7
Barley Protein Extraction and Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Barley protein extractions were done according to the method of Celus et al. [37 (link)], with some modifications: for extraction of albumins and globulins, 50 mg of the sample with 1 mL of 4 M sodium chloride were vortexed for 5 min at 3500 rpm (VibroMix 204 EV, Tehtnica, Slovenia), incubated at 25 °C for 30 min at 500 rpm (Thermomixer 5436, Eppendorf, Germany) and centrifuged (Centrifuge 5415 C, Eppendorf, Germany) for 10 min at 14000 rpm. The supernatant was decanted, filtered in glass vials through 0.45 μm polyvinylidene fluoride (PVDF) membrane filter (Ahlstrom GmbH, Germany) and stored at −20 °C until analysis. Hordeins were extracted from the pellet residue by adding 1.0 mL of 50% 1-propanol + 1% dithiothreitol, vortexed for 5 min, incubated at 60 °C for 60 min, and centrifuged for 15 min. The supernatant was decanted, filtered in glass vials through 0.45 μm PVDF membrane filter and stored at −20 °C until analysis. All extractions were done as duplicates.
8
Dynamic Light Scattering and Rheology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 18
Dynamic light scattering (DLS) was performed using a Malvern Nano Zetasizer ZS (Malvern Instruments Ltd Enigma Business Park, Grovewood Road, Malvern, Worcestershire, UK. WR14 1XZ) and a Haake Rheostress 1 (Thermo Fisher Scientific, Karlsruhe, Germany) equipped with a cone with 60 mm diameter/0.5° angle for buffer viscosity measurements.
All samples were centrifuged (Centrifuge 5415C, Eppendorf, Vienna, Austria) for 5 min at 10.000 rpm to determine the hydrodynamic diameter of a protein. 60 μL of sample were filled into a ZEN0040 disposable micro cuvette and viscosity of buffer was determined by Rheostress 1. This parameter is used for analyzing effective size of proteins by DLS. Operation temperature was 25° C. with an equilibration time of 2 minutes. The proteins angle was set to 173° backscatter to measure the size of and 3 runs per sample were performed to average the results.
Samples were measured by increasing temperature mode to monitor the influence of temperature on a protein. Measurement procedure was similar to a normal size measurement, except for different temperatures with an increasing value of 1° C./min from 15° C. to 80° C. and an equilibration time of 2 min. A DTS2145 low volume glass cuvette was used for these temperature ramps.
9
Plasma/CSF Cefazolin Extraction Protocol
10
Size-Exclusion HPLC Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 17
SE-HPLC was performed using an AKTA Purifier “900-series” (GE Healthcare). The system was equipped with a Superose 12 GL column (GE Healthcare, TC10/30) which was run at a constant flow rate of 0.3 mL per minute at room temperature. As running buffer 20 mM Tris, 100 mM sodium acetate, 500 mM sodium chloride, pH 7.4 was used. The sample was centrifuged (Centrifuge 5415C, Eppendorf, Vienna, Austria) for 5 min at 10,000 rpm and 100 μL were applied automatically by an autosampler. The absorbance of the column effluent was measured continuously at 280 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!