Each BP (1 μg or 0.1 μg AF647-RIS) was complexed with the RALA peptide (1 μg/μL) at a range of mass ratios. For example, to achieve a ratio of 10:1, 10 μg of RALA peptide was added to 1 μg of BP dissolved in TE buffer and the total volume was adjusted to 50 μL with TE buffer (Invitrogen, Thermo Fisher Scientific, UK) pH 8.0 and UltraPure DNase/RNase-Free Distilled Water (Invitrogen, Thermo Fisher Scientific, UK). For RALA/AF647-RIS NPs, the pH was adjusted to 7.0. Samples were left to incubate at RT for 30 min before physiochemical characterisation and functional studies commenced.
Te buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
TE buffer is a commonly used buffer solution for various molecular biology applications. It consists of Tris-HCl and EDTA, providing a stable pH environment and chelating divalent cations. TE buffer is employed for diluting, storing, and stabilizing nucleic acids, such as DNA and RNA, while maintaining their integrity.
Automatically generated - may contain errors
Lab products found in correlation
Assay loading reagent, by Standard BioTools (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Fastprep 24, by MP Biomedicals (2 mentions)
Rnase r, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Dl600, by Keygen Biotech (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Cfx96 touch thermocycler, by Bio-Rad (2 mentions)
Cellsdirect one step qrt pcr kit, by Thermo Fisher Scientific (2 mentions)
Biomark hd, by Standard BioTools (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Hx ifc controller, by Standard BioTools (2 mentions)
Sample loading reagent, by Standard BioTools (2 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Quant ittm picogreendsdna reagent, by Thermo Fisher Scientific (1 mentions)
Repli g single cell kit, by Qiagen (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
73 protocols using te buffer
1
RALA-Mediated Nanoparticle Formulation
2
Quantifying DNA Content in PSC-NPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After washing with PBS to remove any unbound cells, scaffold cultures were placed directly into 1 mL lysis buffer [10 mM Tris pH 8, 1 mM EDTA and 0.2% v/v Triton X-100] within 1.7 mL microtubes. Samples were vortexed for 10 s every 5 min, for a total period of 30 min, and kept on ice between mixes. At this point, samples were either placed in -80 °C for storage or processed further. Samples were then homogenised 10-15 times using a 21gauge needle to produce cell lysate. Lysate was then diluted 1:10 in 1 × TE buffer (Thermo Fisher Scientific). Quant-iT TM PicoGreen® dsDNA reagent (Thermo Fisher Scientific) was diluted 1:200 in 1 × TE buffer. A 1:1 mixture of lysate solution and 1 × PicoGreen® reagent was placed in an OptiPlate-96, black opaque 96-well microplate (PerkinElmer). Fluorescence was measured at excitation and emission wavelengths of 460 nm and 540 nm, respectively, using an EnSpire TM 2300 Multilabel Plate Reader (Perkin Elmer) running EnSpire 3.0 software (Perkin Elmer).
Results were compared to a standard curve for 1-10 × 10 6 hPSC -NPCs analysed as above for dsDNA amount. Human PSC-NPCs cultured on laminin-coated 2D TCPS were harvested via 10 min incubation in a solution of 500 μL Accutase. Human PSC-NPCs were quantified using the trypan blue exclusion method with a 0.4% w/v solution of trypan blue and counted using a hemocytometer.
Results were compared to a standard curve for 1-10 × 10 6 hPSC -NPCs analysed as above for dsDNA amount. Human PSC-NPCs cultured on laminin-coated 2D TCPS were harvested via 10 min incubation in a solution of 500 μL Accutase. Human PSC-NPCs were quantified using the trypan blue exclusion method with a 0.4% w/v solution of trypan blue and counted using a hemocytometer.
3
Single-cell DNA extraction and genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The demi-blastocysts were washed three times in PBS supplemented with 0.02% polyvinyl alcohol (PVA, Sigma-Aldrich), and then washed in PBS without PVA. The
blastocysts were individually collected into 0.2 ml tubes containing 1.5 μl PBS (Invitrogen). DNA extraction and WGA were performed using the REPLI-g Single
Cell Kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions with some modifications. Briefly, 1.5 μl buffer DLB containing dithiothreitol
(built into the kit) was added to the tubes containing a demi-blastocyst, incubated at 65°C for 10 min, and then 1.5 μl Stop Solution (built into the kit) was
added. Next, 20 μl master mix containing DNA polymerase (built into the kit) was added and incubated at 30°C for 8 h. DNA processed for WGA was quantified using
NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), diluted with TE buffer (Invitrogen) to a concentration of 106.8–111.3 ng/μl, and stored at −80°C
until SNP genotyping.
DNA samples were processed for SNP genotyping using the Infinium XT Assay Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. SNP
genotyping was performed using XT Chip (LIAJ custom_50v1, 50,019 SNPs) customized by the Livestock Improvement Association of Japan (Maebashi Institute of
Animal Science, Gunma, Japan).
blastocysts were individually collected into 0.2 ml tubes containing 1.5 μl PBS (Invitrogen). DNA extraction and WGA were performed using the REPLI-g Single
Cell Kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions with some modifications. Briefly, 1.5 μl buffer DLB containing dithiothreitol
(built into the kit) was added to the tubes containing a demi-blastocyst, incubated at 65°C for 10 min, and then 1.5 μl Stop Solution (built into the kit) was
added. Next, 20 μl master mix containing DNA polymerase (built into the kit) was added and incubated at 30°C for 8 h. DNA processed for WGA was quantified using
NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), diluted with TE buffer (Invitrogen) to a concentration of 106.8–111.3 ng/μl, and stored at −80°C
until SNP genotyping.
DNA samples were processed for SNP genotyping using the Infinium XT Assay Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. SNP
genotyping was performed using XT Chip (LIAJ custom_50v1, 50,019 SNPs) customized by the Livestock Improvement Association of Japan (Maebashi Institute of
Animal Science, Gunma, Japan).
4
CD133 Expression Analysis via FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were dissociated from the dish using TE buffer (Invitrogen, Carlsbad, CA, USA) and labeled with an anti-mouse CD133-PE antibody (eBioscience, San Diego, CA, USA) on ice for 30 min following the standard protocol. The cells were analyzed using a FACS Calibur flow cytometer (San Jose, CA, USA). Summit software (FlowJo, Los Angeles, CA, USA) was used to determine the number of positive cells.
5
Single-Cell Multiplex qRT-PCR on Fluidigm Platform
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multiplex qRT–PCR was performed using the Fluidigm (BioMark) platform. TaqMan probes (ThermoFischer) (see Supplementary Data 5 ) were pooled to a concentration of 0.2×. Before cell sorting, an RT-PreAmp master mix (consisting of 5 µl 2× Reaction Buffer and 1.2 µl RT/Taq enzyme (ThermoFischer SuperScript III One-Step RT-PCR System with Platinum Taq kit), 0.1 µl SUPERase-In RNAse Inhibitor (Ambion), 1.2 µl TE buffer (Invitrogen), and 2.5 µl of 0.2× TaqMan assay mix) was freshly prepared on the day. All pipetting steps were performed in a clean-room.
Hundred cells were directly sorted by FACS into 10 µl of RT-PreAmp master mix. Collected samples were immediately vortexed and spun to aid cell lysis. RT-PCR (15 min at 50 °C; 2 min at 95 °C) and pre-amplification (15 s at 95 °C and 4 min at 60 °C; 20 cycles) was performed in a PCR machine and diluted with 40 µl TE buffer. The pre-amplified mix was immediately frozen at −20 °C until further analysis. Gene expression analysis was performed using a 48.48 IFC chip (Fluidigm) according to the manufacturer’s instructions. Ct thresholds were set for each assay with the same thresholds used across all experiments. Ct values were calculated by BioMark Real-time PCR Analysis software (Fluidigm). Data were exported to Excel as.csv files for subsequent analysis.
Hundred cells were directly sorted by FACS into 10 µl of RT-PreAmp master mix. Collected samples were immediately vortexed and spun to aid cell lysis. RT-PCR (15 min at 50 °C; 2 min at 95 °C) and pre-amplification (15 s at 95 °C and 4 min at 60 °C; 20 cycles) was performed in a PCR machine and diluted with 40 µl TE buffer. The pre-amplified mix was immediately frozen at −20 °C until further analysis. Gene expression analysis was performed using a 48.48 IFC chip (Fluidigm) according to the manufacturer’s instructions. Ct thresholds were set for each assay with the same thresholds used across all experiments. Ct values were calculated by BioMark Real-time PCR Analysis software (Fluidigm). Data were exported to Excel as.csv files for subsequent analysis.
6
Quantifying DNA from Papain Digests
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One half of each construct was lyophilized and a dry weight obtained. Samples were incubated in 1.0 ml Papain Solution (2 mM Dithiothreitol and 300 ug/ml Papain) at 60 °C in a water bath for 12 h. A double stranded DNA quantification assay (Quant-iT PicoGreen™ Invitrogen, Carlsbad, CA) was performed. Double stranded DNA extracted from bovine thymus was mixed with TE buffer (Invitrogen, Carlsbad, CA) to create standard DNA concentrations of 1,000, 100, 10, and 1 ng/mL. The standards and 100 uL of each papain digested sample (used in the above GAG and hydroxyproline assays) were added to a black 96 well plate. 100 uL of 2 ug/mL of Pico Green reagent was added to each well and the plate was incubated for 5 min. Sample fluorescence was read at 485 nm excitation/528 nm emission by a spectrophotometric plate reader (Synergy HT–KC-4; BioTek, Winooski, VT). Absorbances were converted to ng/mL concentrations and total double stranded DNA yield in ng using FT4 software (BioTek, Winooski, VT).
7
RT-qPCR Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEC RNA was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) and pre-amplified using pooled TaqMan™ primers (Supplementary Table 1 ) at a concentration of 0.4X and a TaqMan™ PreAmp Master Mix Kit (ThermoFisher). Thermocycling parameters can be found in Supplementary Table 2A . Pre-amplified cDNA samples were diluted 1:20 in 1X TE buffer (Invitrogen) and combined in the appropriate ratio with TaqMan™ Gene Expression Master Mix (ThermoFisher), nuclease-free water, and the appropriate primer on a MicroAmp Fast Optical 96-Well Reaction Plate (0.1, ThermoFisher). Samples were assayed in triplicate for each primer and RT-qPCR was performed using a StepOnePlus™ Real-Time PCR System (ThermoFisher). Thermocycling parameters are can be found in Supplementary Table 2B . The data obtained were normalised by subtracting the ΔCT, value averaged across triplicates, for the reference gene (Pgk1) from that of the target gene for test and control groups.
8
Endothelial Cell Culture and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human umbilical vein endothelial cells (HUVECs) and immortal human endothelial cells (EA.hy926) were obtained from American Type Culture Collection (ATCC, Manassas, VA). The EGM-2 Bullet kit and HEPES-buffered saline solution, to culture HUVEC cells, were from Lonza (Walkersville, MD). EA.hy926 cell were maintained in Dulbecco’s Modified Eagle Medium (DMEM). Heat-inactivated Fetal Bovine Serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA). To dislodge adherent cells 0.05% trypsin was obtained from HyClone (Pittsburgh, PA). RIPA cell lysis buffer, Tris-Glysine SDS gel running buffer, transfer buffer, and laemmli SDS 4X sample buffer were procured from Boston BioProducts (Ashland, MA). Recombinant Hirudin from yeast was obtained from American Diagnostica Inc. (Stamford, CT). Chromogenix S-2366 was obtained from DiaPharma Group Inc. (West Chester, OH). Primary TM antibody was procured from Novus Biologicals (Catalogue number: NBP1-95319; Littleton, CO) and goat anti-rabbit IgG-HRP conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Catalogue number: SC-2004; Santa Cruz, CA). TE buffer and DPBS were obtained from Invitrogen (Grand Island, NY). GT3 was procured from Yasoo Health Inc. (Johnson City, TN).
9
Collecting Subgingival Biofilm Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supragingival biofilm was removed with sterile gauze and subgingival biofilm samples were collected from the mesial buccal or mesial vestibular of the first or second molar by using two sterilized paper points (No. 30 Tanari, Tanariman Ind Ltd, Manacapuru, AM, Brazil) inserted into gingival crevice for 30 seconds. Twenty-seven subgingival samples from nine women during the 2nd (9 samples) and 3rd (9 samples) semesters of pregnancy and from nine non-pregnant women (9 samples) were collected. The paper points were transferred into a tube containing 300 µl of TE buffer (Invitrogen, Invitrogen do Brasil Ltd, São Paulo, SP, Brazil), and stored at -80°C.
10
cDNA Synthesis with GrandScript Kit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reverse transcription was performed using GrandScript cDNA Synthesis kit (TATAA Biocenter) in 10 μL reactions, including 2 μL 5× GrandScript RT reaction mix, 0.5 μL GrandScript RT enzyme, 5.5 μL water and 2 μL of lysate. RT was performed at 25 °C for 5 min, 42 °C for 30 min and terminated at 85 °C for 5 min. Samples were diluted with 50 μL TE buffer, pH 8.0 (Invitrogen) and stored −20 °C before downstream qPCR.
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!