CD4+ T cell surface biotinylation using pre-complexed Siglec-10 Fc and Protein A-APEX2
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Olivier Julien Matthew Macauley
PROVIDER: MSV000100018 | MassIVE | Tue Nov 25 11:20:00 GMT 2025
SECONDARY ACCESSION(S): PXD071203
REPOSITORIES: MassIVE
Similar Datasets
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Genistein in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 10pM (very low, vL), 1 nM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of Genistein. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at time each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing.
2016-07-22 | GSE71717 | GEO
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Bisphenol A in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 1pM (very low, vL), 100pM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of BPA. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing. Keywords: Dose Response/Time Course
2009-08-13 | GSE17624 | GEO
Project description:In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells. Cell culture conditions: iNdiPSCs were grown on feeder cells in 2i medium (Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO), 1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA), 1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]). Virus production and infection: Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium: DMEM/F-12 supplemented with B27 (both GIBCO), 10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG. Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium. Alkaline phosphatase staining Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit (product no. F4648) for 20 min in the dark at RT. Chromosome spread: Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl, incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative (3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT. Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide. Drops were air dried for 30 min and mounted with DAPI containing mounting solution. RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR) RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad). Oct4 promoter methylation analysis: Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104). Followed by nested PCR using a set of target region specific primers. The first PCR was performed using 1micro-l of bisulfite converted and purified DNA, SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s, followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each. For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s, followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C, and finally an additional elongation step for 5 min at 68C. The amplified fragments were resolved on a 1.5% agarose gel, purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen). The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates. The picked colonies were sent to GATC for sequencing. The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp). Immunocytochemistry: Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed again twice in PBS. Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT. The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash. The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000 anti SSEA1, (MC-480, Millipore), 1:160, anti-3-Tubulin (T8660, Sigma), 1:2000, anti-Smooth muscle actin (M0851, DAKO), 1:200, anti-Sox17 (AF1924, R&D Systems), 1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000. DNA extraction and PCR: DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306). PCR was carried out using Taq polymerase including buffers from New England Biolabs, following their recommended protocol. The following primers were used for genotyping: pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4. In vitro differentiation: First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted. After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG), which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above. Teratoma formation: 1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA. Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin. Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute. Microarray analysis: Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides. Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.
2014-03-28 | GSE44284 | GEO
Project description:Overnight cultures of S. aureus were diluted to OD600 = 1 and 1 ml of culture was resuspended in 500 μl of either PBS, human plasma, or human serum, and incubated rotating at 37 °C for 30 m. Suspensions were washed 3x with PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide. Cells were incubated with immobilized trypsin (ThermoFisher 20230), suspended in PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide, at 37 °C for 2 h. cells were pelleted, and the supernatant containing protein fragments was analyzed by mass spectrometry to determine proteins.
2023-04-14 | PXD038927 | Pride
Project description:DO11.10 x TCR-Calpha-/- T cells stimulated under Th2 conditions for 6 days were centrifuged over Histopaque 1083 (Sigma) to remove dead cells, washed and rested for 20 min at 37C in complete media with 0.1 mg/ml cycloheximide (CHX, Sigma) added for the final 10 min. Restimulated cells were activated with 10 ug/ml plate-bound I-Ad/ova complexes for 3 hrs. Cells were washed with ice-cold PBS/CHX and resuspended in polysome extraction buffer [30 mM HEPES (pH 7.4), 15 mM MgCl2, 0.3 M NaCl, 1% Triton X-100, 0.1 mg/ml CHX, 1 mg/ml heparin, 500 U/ml SUPERase-In RNAse inhibitors (Ambion, Austin, TX), 1x Complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN)]. After incubation on ice for 25 min, nuclei and debris were removed by centrifuging and the cleared supernatants transferred to fresh tubes. 1.5 ml of each extract (from 3 x 10exp8 T cells) were layered on a 37.5 ml 10-50% sucrose gradient prepared in SW28 ultraclear centrifuge tubes (Beckman Coulter, Fullerton, CA) using the salts described above, but without detergent, RNAsin or protease inhibitors. After centrifugation for 220 min at 28,000 rpm (average RCF = 103,745), fractions were isolated using an ISCO density gradient fractionator (Lincoln, NE) by pumping 60% sucrose into the bottom of the tube and collecting the displaced volumes into acid phenol:chloroform, with constant monitoring of ultraviolet absorbance at 254 nm. Extracted RNA was precipitated with isopropanol, washed and resuspended in distilled water. RNA from polysome associated fractions 7-12 of the gradients described above were pooled and 30-100 ug of total RNA per sample were used to template a Superscript III reverse transcription reaction (Invitrogen, Carlsbad, CA) incorporating amino-allyl dUTP (Sigma). cDNA samples were washed with distilled water on microcon-30 columns (Millipore, Billerica, MA), dried and labeled with Cy3 (primed samples) or Cy5 (restimulated samples) using CyDye reactive dye labeling kits (Amersham). Unincorporated dye was removed using QiaQuick PCR purification columns (Qiagen), labeled cDNAs were eluted in 10mM Tris, pH 8, combined and dried. Fluorescence in the Cy3 and Cy5 channels was acquired using an Axon 4000B microarray scanner and GenePix software (Axon Instruments, Union City, CA). Data were normalized using the R package Bioconductor software and lowess normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996). Keywords = Th2 differentiation Keywords = translation Keywords = polysome profile Keywords: cell stimulation
2006-03-23 | GSE4500 | GEO
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1031 | GEO
Project description:In mammals, the peptide hormone vasopressin controls renal water excretion, largely through its actions in the renal collecting duct to regulate the molecular water channel aquaporin-2 (AQP2). There are two modes of regulation: 1) short-term regulation by membrane trafficking; and 2) long-term regulation involving vasopressin-induced changes in the abundance of the aquaporin-2 protein. Defects in the long-term regulation have been implicated in multiple water balance disorders. With the objetive to identify genes that are transcriptionally regulated in response to vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq to measure all transcripts (n=9) and ChIP-seq for RNA polymerase II (Polr2a) binding along the genome (n=3). Observations were made both after 24-hr treatment with the vasopressin analog dDAVP and with vehicle. Protocol:Confluent monolayers mpkCCD cells were washed in ice-cold 1X PBS (Ca+2/Mg+2-free) followed by cross-linking in 1.11% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5min at room temperature with gentle rocking (PN 520075, Covaris, Woburn, Massachusetts). Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and 1X ice-cold PBS was added to each plate. Cells were scraped into 15ml conical tubes, spun down (200 x g, 5 min), and washed twice with PBS. After the second wash, cell pellets were resuspended in 10 ml of 1X Covaris Lysis Buffer for 10 min at 4o C with rocking. Nuclei were pelleted by spinning at 1,700 x g for 5 min at 4o C. Nuclear pellets was resuspended in the 1X Covaris Wash Buffer and incubated for 10 mins at 4o C with rocking. Samples were spun at 1,700 x g for 5 min at 4o C. Nuclear pellets were washed twice with the Covaris Non-ionic Shearing Buffer and spun at 1,700 x g for 5 min at 4o C. Nuclei were resuspended in Covaris Non-ionic Shearing Buffer (Maximum of 1-3x 10^7cells per ml of buffer). Shearing. Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm) and volumes adjusted to 1 ml (V)). Samples were sheared using a Covaris S2 SonoLAB Single system for two minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mcg/ml (R) was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). To test shearing efficiency, a 20 ul aliquot from each tube was removed after shearing to test the efficacy of shearing. The aliquots, including the unsheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 mins at 37o C. 1 ul proteinase K (20 ug/ul) was added and the samples were incubated overnight at 65o C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-700 bp in length with average fragment size of 300 bp. Testing epitope integrity (immunoblotting). Total protein content was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Samples were then diluted with sample buffer (5X Laemmli buffer) at 20% (v/v) of the entire sample volume. Proteins were resolved by SDS-PAGE (4-20% polyacrylamide gels, Criterion, Bio-Rad, Hercules, CA) and transferred electrophoretically onto nitrocellulose membranes as previously described (Hwang, 2010). Membranes were probed with anti-RNA polymerase II (Catalog #: MMS-126R, Covance). The antibody was used at a 1:500 dilution (anti-RNA polymerase II) (in Odyssey blocking buffer containing 0.1% Tween 20) overnight at 4°C. After 1-h incubation with secondary antibody (Li-Cor Biosciences 680 anti-rabbit immunoglobulin G or 800 anti-mouse immunoglobulin G; Lincoln, NE) at 1:5000 dilution, sites of antibody-antigen reaction were detected using an Odyssey infrared imager (Li-Cor). Chromatin immunoprecipitation. 10 ul of sample equivalent to 2% input was removed prior to IP. Chromatin Immunoprecipitation was performed using 30-90 ug of sheared chromatin in 500 uL of 1X Covaris Non-ionic Shearing Buffer with 10 μl anti-RNA polymerase II antibodies. Samples were incubated overnight at 4°C with rotation, followed by addition of ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) and incubation for 2 h at 4o C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 mins/wash at 4o C (SimpleChIP protocol #9003, Cell Signaling Technology). Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 mins at 65o C in a thermomixer. To reverse crosslinks, 6 ul of 5M NaCl and 2 ul of 20 ug/ul Proteinase K were added to the chromatin followed by incubation at 65o C overnight. DNA was purified according to SimpleChIP protocol #9003, Cell Signaling Technology. q-PCR. To check the success of the chromatin immunoprecipitation, input and ChIP’ed DNA samples were amplified with SYBR Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System. Primer sets corresponding to Hox9a and actin genes were used as positive controls for both histone and RNA polymerase II IP’s. (see Supplementary Table X for primer sequences). Library construction, amplification and sequencing. Loaded fixed volume of total eluate. Library construction was performed on a NuGEN instrument using the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit. Libraries were then enriched via PCR. After amplification, DNA was measured. A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data. High-throughput sequencing and read mapping. Libraries for Illumina sequencing of the samples were prepared using the the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit , NuGen Mondrian SP+ (NuGEN, San Carlos, CA) . Samples were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads.
2016-07-01 | GSE79573 | GEO
Project description:Preparation of AMDs. 125 μM Ac4ManNAz were incubated with Raw 264.7 (~ 107 cells/dish) for 24, 48, and 72 h, followed by gentle washing with PBS or complete medium to produce Raw 264.7-Ac4ManNAz. Subsequently, Raw 264.7-Ac4ManNAz (~ 107 cells) were resuspended in PBS containing Alkynyl-Ce6(0.1 μM), ascorbic acid (0.2 μM) and CuSO4 (0.2 μM) and incubated for 1h at 37 °C. Then centrifuged (1000 rpm, 3 min) and washed 3 times with PBS to obtain precursor of AMDs. precursor of AMDs (~ 107 cells/tube, 1.0 ml) was immersed in liquid nitrogen, 24 h later, the frozen tubes were thawed at 37°C, centrifuged at 1500 rpm for 3 min, washed and resuspended by PBS to generate the AMDs (5 x 106 units/tube, 1.0 ml).
2024-03-21 | PXD050063 | Pride
Project description:Colorectal cancer tissue sections were obtained according to the inclusion criteria. The formalin was used to immersed all cancer specimens. And tissues were cut to 5 μm thickness and placed on glass slides before staining. Endogenous peroxidase activity was inhibited and blocked by de-paraffinizing, rehydrating, and using 5% bovine serum albumin at 37ºC for 30 min. The treated sections were incubated with anti-FOS (promab 30360) at 4ºC overnight and washed three times with PBS. After that, it is required that incubation with secondary anti-peroxidation sunflower at 37ºC for 30 minutes. After washing three times again with PBS, the sections were developed in diaminobenzidine and microscopic images were made by light microscopy.
| 6033 | ecrin-mdr-crc