HEK293T cells were transfected by packaging plasmids, psPAX2 (Addgene Cat#12260) and pMD2.G (Addgene Cat#12259), and a plasmid of gene of interest in lentiviral backbone (pLenti-CAG-mCherry, pLenti-CAG-NOTCh2NLB-ires-EGFP, pLenti-CAG-NOTCH2NL-ΔEGF-ires-EGFP, pLenti-CAG-NOTCH2NL-ΔC-ires-EGFP and pLenti-CAG-NICD-ires-EGFP). 2 days after transfection, culture medium was collected and viral particles were enriched by filter device (Amicon Ultra-15 Centrifuge Filters, Merck, Cat#UFC910008). Titer check was performed on HEK293T cell culture for every batch of lentiviral preparation.
Amicon ultra 15 centrifuge filter
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra-15 Centrifuge Filters are a type of laboratory equipment used for sample preparation and purification. They are designed to separate and concentrate macromolecules such as proteins, nucleic acids, and other biomolecules from complex mixtures through a centrifugation process.
Automatically generated - may contain errors
10 protocols using amicon ultra 15 centrifuge filter
1
Lentiviral Packaging and Titration
2
Glycation of Model Protein Myoglobin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The glycation products used in this study were generated as described earlier using either conventional glycation in solution (ACG) or reaction under dry conditions (MWG) [23 (link)]. Briefly, the MiliQ water solution mixtures of a model protein MB and melibiose (mel) in a 1:100 molar ratio (protein:carbohydrate) were prepared, followed by the ACG or MWG reaction. In the ACG method, the MB/mel solution was incubated for 21 days at room temperature, while in the MWG method, an analogous MB/mel solution was lyophilized and incubated in a microwave initiator reactor (Biotage, Uppsala, Sweden) for 45 min at 85 °C. Next, the samples were dissolved in MilliQ water and centrifuged at 5000× g for 15 min to remove any insoluble precipitates. The obtained supernatants were dialyzed against MilliQ water on Amicon Ultra-15 centrifuge filters (Merck Millipore, Burlington, MA, USA) with a 30 kDa cut-off to separate unreacted substrates and MB monomers. The resulting glycation products were lyophilized and stored at −20 °C for further characterization.
3
Ovalbumin Hydrolysis with Artichoke Extract
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hydrolysis of commercial ovalbumin (Acros Organics) with the artichoke extract was developed using the optimal conditions previously studied [15 ]. For this, an ovalbumin solution of 1% (w/v) in distilled water at a pH of 6.2 was prepared. The reaction was performed in tubes submerged in a shaking bath at 36 °C with an enzyme:substrate ratio of 0.0232.
Ovalbumin hydrolysates were obtained at different hydrolysis times, 2 (OH2), 4 (OH4), and 16 (OH16) h, to evaluate the effect of the hydrolysis time in obtaining peptides with bioactivity. The reaction was stopped at the corresponding time in each case, adjusting the pH to 4.5 with HCl, the isoelectric point of ovalbumin that causes its precipitation, then, it was centrifuged for 20 min at 4000× g and filtered with a 0.45 µm nylon filter. Finally, the hydrolysate obtained was raised to a pH of 7 with NaOH, dispensed in Falcon tubes and reserved at −20 °C until its utilization. Each type of hydrolysate was obtained in triplicate.
Furthermore, molecular weight fractions < 3 kDa were obtained from each of the hydrolysates. For this, ultrafiltration was performed by centrifugation with Amicon Ultra-15 centrifuge filters of regenerated cellulose (3000 NML; Merck Millipore, Burlington, MA, USA) at 4 °C and at 4000× g for 40 min. The permeate was collected in Falcon tubes at −20 °C until use.
Ovalbumin hydrolysates were obtained at different hydrolysis times, 2 (OH2), 4 (OH4), and 16 (OH16) h, to evaluate the effect of the hydrolysis time in obtaining peptides with bioactivity. The reaction was stopped at the corresponding time in each case, adjusting the pH to 4.5 with HCl, the isoelectric point of ovalbumin that causes its precipitation, then, it was centrifuged for 20 min at 4000× g and filtered with a 0.45 µm nylon filter. Finally, the hydrolysate obtained was raised to a pH of 7 with NaOH, dispensed in Falcon tubes and reserved at −20 °C until its utilization. Each type of hydrolysate was obtained in triplicate.
Furthermore, molecular weight fractions < 3 kDa were obtained from each of the hydrolysates. For this, ultrafiltration was performed by centrifugation with Amicon Ultra-15 centrifuge filters of regenerated cellulose (3000 NML; Merck Millipore, Burlington, MA, USA) at 4 °C and at 4000× g for 40 min. The permeate was collected in Falcon tubes at −20 °C until use.
4
Amplification and Cloning of APCDD1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length APCDD1 sequence was amplified from the cDNA of 293T cells and cloned into the PCDH-CAG-IRES-mCherry backbone (from the laboratory of B. Hu). Lentiviruses were produced by transfecting 293T cells using the PCDH-APCDD1-IRES-mCherry plasmid or PCDH-IRES-mCherry plasmid combined with the packaging plasmids psPAX2 and pMD2.G. Then, the supernatant was collected for 3 days after transfection and concentrated with a filter device (Amicon Ultra15 Centrifuge Filters, Merck, Cat# UFC910008).
5
Generation and Characterization of 4T1 Breast Cancer Cell Supernatant
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine 4T1 breast cancer 4T1 cells were obtained from (ATCC, Manassas, VA, USA) and grown in RPMI 1640 supplemented by 10% FBS, 100 μM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1 mM sodium pyruvate (complete medium), as previously described [12 (link)]. The generation of GM-CSF-deficient 4T1 cells (A8) was previously reported [13 (link)]. To generate 4T1 cell culture supernatant (4T1-sup), 4T1 cells were grown in the complete medium in T-75 tissue culture flasks. When the color of the media turned slightly yellow, cell-free supernatants were collected by centrifugation at 1200 rpm for 10 min. Cell-free supernatants were concentrated by using Amicon Ultra-15 Centrifuge filters (Merck Millipore Ltd., Cork, Ireland).
6
Lentiviral Transduction for Cortical Differentiation and Organoid Analyses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were transfected by packaging plasmids, psPAX2 (Addgene) and pMD2.G (Addgene), and the transfer vector using Xtreme gene 9 transfection reagent (Sigma-Aldrich). Two days after transfection, culture medium was collected and viral particles were enriched by filtration (Amicon Ultra-15 Centrifuge Filters, Merck). Titration of every batch of lentiviral preparation was estimated following transduction of HEK293T cells. For 2D cortical differentiations, the hES cell-derived cortical cells were infected by the lentiviral constructs at differentiation day 25. The culture medium was changed the next day and phenotypes were analyzed 6 days after infection for CROCC & CROCCP2 subcellular localisation. For infection of cortical organoids, lentiviral preparations were ultracentrifugated at 100,000 x g, 4°C for 2 h and pellets resuspended in sterile PBS in order to prevent cell toxicity. Mouse primary cortex cultures were infected on the day of infection and phenotypes were analyzed 72h later.
7
Lentiviral Infection of Cortical Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were transfected by packaging plasmids, psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259), and the transfer vector using Xtreme gene 9 transfection reagent (Sigma-Aldrich). Two days after transfection, culture medium was collected and viral particles were enriched by filtration (Amicon Ultra-15 Centrifuge Filters, Merck). Titration of every batch of lentiviral preparation was estimated following transduction of HEK293T cells. The hES cell-derived cortical cells were infected by the lentiviral constructs at differentiation day 25. The culture medium was changed the next day and phenotypes were analyzed 6 days after infection for CROCC & CROCCP2 subcellular localisation. Mouse primary cortex cultures were infected on the day of infection and phenotypes were analyzed 72h later.
8
Bioactive Casein Hydrolysates: Optimizing Time
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the effect of hydrolysis time on bioactivity, we obtained 3 casein hydrolysates, at 2 (CH2), 4 (CH4), and 16 (CH16) h. Each was obtained 3 times under the previously established optimal hydrolysis conditions (Bueno-Gavilá, 2017) . A solution of commercial total bovine casein (carbohydrate-and fatty acid-free; Calbiochem, Calbiochem EMD Chemicals Inc., San Diego, CA) was prepared at 1% (wt/vol) in distilled water, adjusting the pH to 6.2 with 1 M NaOH. The hydrolysis reaction occurred at 50°C at a concentration of enzymatic protein extract of 23 µg/ mL of casein solution. The reaction was stopped at 2, 4, or 16 h by increasing the temperature to 100°C for 10 min, and the pH was adjusted to 4.6 with HCl (1 M). Then, the hydrolysates were centrifuged at 4,000 × g for 20 min, and the supernatant was collected and filtered through a 0.45-µm nylon filter. Finally, the pH was adjusted to 7 with NaOH (1 M) and distributed into Falcon tubes for storage at -20°C until use. To obtain the molecular weight fraction <3 kDa, we used Amicon Ultra-15 centrifuge filters of regenerated cellulose (3.000 NMWL; Merck Millipore, Burlington, MA). The filtration was carried out by centrifuge in refrigerated conditions at 4°C at 4,000 × g for 40 min. The resulting permeate was stored in Falcon tubes and frozen at -20°C until use.
9
Micro-emulsion Synthesis of PM6 Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PM6 5 mg was dissolved in 1mL chloroform and stirred at 50 °C for 3 h. Then, the solution was added to 10 mL 10 mg mL−1 DIMMBr aqueous solution and stirred at 40 °C for 1 h. The formed micro-emulsion dispersion was ultrasonicated using a SCIENTZ-IID ultrasonic finger (Scientz Biotechnology Co., Ltd., Ningbo, China) in a water bath. The mini-emulsion system was constantly stirred until chloroform was completely eliminated. The excess surfactant from the particle solution was removed using an Amicon® ultra-15 centrifuge filter (cutoff 30 K) (Sigma Aldrich, St. Louise, MO, USA). The dispersion was placed into the filter and centrifuged at 4000 rpm for 20 min. The retentate was raised to 15 mL with water and then centrifuged again. This process was repeated several times until the surface tension of the filtrate reached 38 ± 2 mN m−1. The retentate was filtered with a 0.45 μm filter before the last centrifugation.
10
Purification of T-AgNPs Exosomes
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!