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Cationic Protein, Eosinophil
Cationic Protein, Eosinophil
Cationic Proteins and Eosinophils: Exploring the Intersection of Immune Regulation and Cellular Function.
Cationic proteins, such as those found in eosinophils, play a crucial role in the body's immune response and inflammatory processes.
These positively charged molecules interact with various cellular targets, modulating immune cell activation and effector functions.
Eosinophils, a specialized type of white blood cell, are a primary source of cationic proteins and are implicated in a range of physiological and pathological conditions, including allergic reactions, parasitic infections, and certain autoimmune disorders.
Understanding the intricate relationship between cationic proteins and eosinophils is essential for advancing research and developing effective therapies targeting these important components of the immune system.
Cationic proteins, such as those found in eosinophils, play a crucial role in the body's immune response and inflammatory processes.
These positively charged molecules interact with various cellular targets, modulating immune cell activation and effector functions.
Eosinophils, a specialized type of white blood cell, are a primary source of cationic proteins and are implicated in a range of physiological and pathological conditions, including allergic reactions, parasitic infections, and certain autoimmune disorders.
Understanding the intricate relationship between cationic proteins and eosinophils is essential for advancing research and developing effective therapies targeting these important components of the immune system.
Most cited protocols related to «Cationic Protein, Eosinophil»
Cationic Protein, Eosinophil
Mus
m6A MeRIP is based on the previously described m6A-seq protocol [27 (link)] with several modifications: 30 μl of protein A magnetic beads (10002D; Thermo Fisher Scientific) and 30 μl of protein G magnetic beads (10004D; Thermo Fisher Scientific) were washed twice by IP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), resuspended in 500 μl of IP buffer, and tumbled with 5 μg anti-m6A antibody at 4 °C for at least 6 hours. Following 2 washes in IP buffer, the antibody–bead mixture was resuspended in 500 μl of the IP reaction mixture containing fragmented total RNA, 100 μl of 5× IP buffer, and 5 μl of RNasin Plus RNase Inhibitor (N2611; Promega, Madison, WI) and incubated for 2 hours at 4 °C.
In the low/high salt-washing method, the RNA reaction mixture was washed twice in 1,000 μl of IP buffer, twice in 1,000 μl of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), and twice in 1,000 μl of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O) for 10 minutes each at 4 °C. After extensive washing, the m6A-enriched fragmented RNA was eluted from the beads in 200 μl of RLT buffer supplied in RNeasy Mini Kit (74106; QIAGEN; Germany) for 2 minutes at room temperature. A magnetic separation rack was used to pull beads to the side of the tube. Supernatant was collected to a new tube, and 400 μl of 100% ethanol was added to it. The mixture was transferred to an RNeasy MiniElute spin column and centrifuged at >12,000 rpm at 4 °C for 1 minute. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 °C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O. For a second round of IP, eluted RNA was re-incubated with protein A/G magnetic beads coupled to anti-m6A antibody, followed by washes, elution from the protein A/G beads, and purification as above. In addition, it is the high salt that contributes to better S/N ratio (S7A Fig ).
In the m6A competitive elution method, the m6A competitive elution buffer for each pulldown was prepared by mixing 45 μl of 5× IP buffer, 75 μl of 20 mM m6A (M2780; Sigma-Aldrich), 3.5 μl of RNasin Plus RNase Inhibitor, and 101.5 μl of ultrapure H2O. The immunoprecipitated m6A RNA with protein A/G magnetic beads was then washed 3 times in 1,000 μl of IP buffer for 10 minutes each at 4 °C and was resuspended in 100 μl of m6A competitive elution buffer with continuous shaking for 1 hour at 4 °C. The mixture was placed on a magnetic separation rack, and supernatant containing the eluted m6A RNA was collected to a new tube. Then, another 100 μl of m6A competitive elution buffer was added for one more elution. To purify the eluted RNA, 700 μl of RLT buffer and 1,400 μl of 100% ethanol were added to 200 μl of eluted supernatant collected and mixed thoroughly. The mixture was transferred to an RNeasy MiniElute spin column (QIAGEN) and centrifuged at >12,000 rpm at 4 °C for 1 minute. This step was repeated until all of the sample was loaded to the column. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 °C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O.
The MeRIP-seq data has been deposited in NCBI GEO database (GSE116002).
In the low/high salt-washing method, the RNA reaction mixture was washed twice in 1,000 μl of IP buffer, twice in 1,000 μl of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), and twice in 1,000 μl of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O) for 10 minutes each at 4 °C. After extensive washing, the m6A-enriched fragmented RNA was eluted from the beads in 200 μl of RLT buffer supplied in RNeasy Mini Kit (74106; QIAGEN; Germany) for 2 minutes at room temperature. A magnetic separation rack was used to pull beads to the side of the tube. Supernatant was collected to a new tube, and 400 μl of 100% ethanol was added to it. The mixture was transferred to an RNeasy MiniElute spin column and centrifuged at >12,000 rpm at 4 °C for 1 minute. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 °C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O. For a second round of IP, eluted RNA was re-incubated with protein A/G magnetic beads coupled to anti-m6A antibody, followed by washes, elution from the protein A/G beads, and purification as above. In addition, it is the high salt that contributes to better S/N ratio (
In the m6A competitive elution method, the m6A competitive elution buffer for each pulldown was prepared by mixing 45 μl of 5× IP buffer, 75 μl of 20 mM m6A (M2780; Sigma-Aldrich), 3.5 μl of RNasin Plus RNase Inhibitor, and 101.5 μl of ultrapure H2O. The immunoprecipitated m6A RNA with protein A/G magnetic beads was then washed 3 times in 1,000 μl of IP buffer for 10 minutes each at 4 °C and was resuspended in 100 μl of m6A competitive elution buffer with continuous shaking for 1 hour at 4 °C. The mixture was placed on a magnetic separation rack, and supernatant containing the eluted m6A RNA was collected to a new tube. Then, another 100 μl of m6A competitive elution buffer was added for one more elution. To purify the eluted RNA, 700 μl of RLT buffer and 1,400 μl of 100% ethanol were added to 200 μl of eluted supernatant collected and mixed thoroughly. The mixture was transferred to an RNeasy MiniElute spin column (QIAGEN) and centrifuged at >12,000 rpm at 4 °C for 1 minute. This step was repeated until all of the sample was loaded to the column. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 °C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O.
The MeRIP-seq data has been deposited in NCBI GEO database (GSE116002).
angiogenin
Antibodies, Anti-Idiotypic
Buffers
Cationic Protein, Eosinophil
Ethanol
G-substrate
GTP-Binding Proteins
Igepal CA-630
Immunoglobulins
Promega
Sodium Chloride
Staphylococcal Protein A
Tissue, Membrane
Tromethamine
DNA was extracted in triplicate from each faecal sample using the five commercial kits (Table 1 ) according to their respective manufacturer's instructions, with slight modifications. Briefly, genomic DNA was extracted using two starting amounts of faecal material, 100 mg and 200 mg. Mechanical cell lysis (bead-beating) was carried out for all kits at 50 Hz for 5 min using the TissueLyser LT™ (Qiagen, FRITSCH GmbH, Idar-Oberstein, Germany), with the exception of QIAamp® DNA Stool Mini Kit (kit QA), seeing that a mechanical cell lysis step is not recommended by the manufacturer.
In order to ensure complete flow-through of supernatant during step 4 for kit Z, an additional centrifugation step at 14 000 rpm for 1 min was added.
Homogenisation of faecal samples, in Buffer ASL, for kit QA was performed using the TissueLyser LT™ (Qiagen, FRITSCH GmbH, Idar-Oberstein, Germany) at 50 Hz for 1 min. The optional heating of the faecal lysate at 95 °C (in step 4) was used instead of 75 °C. When required, the alternate RNase treatment, as recommended by the manufacturer was used. Extracted DNA was treated with 3.0 μg of RNase A (Sigma-Aldrich, Carlsbad, United States of America). Because the manufacturer's protocol did not include a subsequent clean-up step, in order to remove the degraded RNA and enzyme from the treated DNA, the wash steps in the protocol were performed from step 11, excluding the incubation step at step 12.
Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P) extractions included the alternate lysis step, as per manufacturer's recommendations, which replaced step 5 for kit P and step 6 for kit U.
All extracted DNA was eluted in 50 μl of distilled water (H2O), except the automated kit QS, where the minimum elution volume allowed by the supplier was set at 60 μl using Buffer AVE.
The five commercial DNA extraction and purification kits used in this study.
| Full name of the kit | Manufacturer details | Kit name abbreviation | Recommended faecal starting amount (mg) | Extraction method | Elution volume (μl) | Estimated price per kit (June 2012, ZAR) |
|---|---|---|---|---|---|---|
| QIAsymphony® Virus/Bacteria Midi Kit | Qiagen, Hilden, Germany | QS | Not specified | Automated | 60, 85, 110 | 4731 |
| ZR Fecal DNA MiniPrep™ | Zymo Research Corp., Irvine, USA | Z | 150 | Manual | 100 | 2128 |
| QIAamp® DNA Stool Mini Kit | Qiagen, Valencia, CA, USA | QA | 180–220 | Manual | 200 | 2297 |
| Ultraclean® Fecal DNA Isolation Kit | MoBio Laboratories Inc., Carlsbad, USA | U | 250 | Manual | 50 | 2530 |
| PowerSoil® DNA Isolation Kit | MoBio Laboratories Inc., Carlsbad, USA | P | 250 | Manual | 100 | 2824 |
Kit QS provides the option of three elution volumes (µl).
In order to ensure complete flow-through of supernatant during step 4 for kit Z, an additional centrifugation step at 14 000 rpm for 1 min was added.
Homogenisation of faecal samples, in Buffer ASL, for kit QA was performed using the TissueLyser LT™ (Qiagen, FRITSCH GmbH, Idar-Oberstein, Germany) at 50 Hz for 1 min. The optional heating of the faecal lysate at 95 °C (in step 4) was used instead of 75 °C. When required, the alternate RNase treatment, as recommended by the manufacturer was used. Extracted DNA was treated with 3.0 μg of RNase A (Sigma-Aldrich, Carlsbad, United States of America). Because the manufacturer's protocol did not include a subsequent clean-up step, in order to remove the degraded RNA and enzyme from the treated DNA, the wash steps in the protocol were performed from step 11, excluding the incubation step at step 12.
Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P) extractions included the alternate lysis step, as per manufacturer's recommendations, which replaced step 5 for kit P and step 6 for kit U.
All extracted DNA was eluted in 50 μl of distilled water (H2O), except the automated kit QS, where the minimum elution volume allowed by the supplier was set at 60 μl using Buffer AVE.
Bacteria
Buffers
Cationic Protein, Eosinophil
Cells
Centrifugation
Enzymes
Feces
Genome
isolation
Ribonuclease, Pancreatic
Virus
Following inactivation, RNA from one aliquot per condition per virus isolate and negative control was immediately extracted with the QIAamp Viral RNA Mini Kit (Qiagen) and stored at −80 °C until further processing. Real-time RT-PCR assays for SARS-CoV-2 RNA detection were performed in duplicate using the Charité Virologie algorithm (Berlin, Germany) to detect both E and RdRp genes [9 (link)]. In brief, real-time RT-PCR was performed using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) on the CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) ABI. Each 25 µl reaction mixture contained 5 µl of RNA, 3.1 µl of RNase-free water, 12.5 µl of 2X PCR buffer, 1 µl of SuperScriptTM III RT/Platinum Taq Mix, 0.5 ul of each 10 µM forward and reverse primer, and 0.25 µl of probe (E_Sarbeco_P1 or RdRP_SARSr-P2) using the following thermal cycling conditions; 10 min at 55 °C for reverse transcription, 3 min at 94 °C for PCR initial activation, and 45 cycles of 15 s at 94 °C and 30 s at 58 °C.
Biological Assay
Buffers
Cationic Protein, Eosinophil
DNA Polymerase III
Genes
Oligonucleotide Primers
Platinum
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Reverse Transcription
RNA, Viral
SARS-CoV-2
Touch
Virus Diseases
cDNA synthesis was essentially performed as previously described
[10 (link)]. Briefly, single tube poly (A) synthesis and reverse transcription was performed with 100 ng of RNA in a final volume of 10 μl combined with 1 μl MuLV reverse transcriptase (New England Biolabs), 1 μl of poly (A) polymerase (New England Biolabs), 1 μl 10x poly (A) polymerase buffer, 1 μl 0.1 mM ATP, 1 μl 10 μM RT-primer (5′-CAGGTCCAGTTTTTTTTTTTTTTTVN where V is A, C and G and N is A, C, G and T). The reaction was incubated at 42°C for 1 hour followed by enzyme inactivation at 95°C for 5 minutes. A panel of three technical cDNA replicates for each of the three biological samples per condition was constructed. The cDNA was diluted 5× before used in qPCR reactions.
miR-specific qPCR was performed as previously described in
[16 (link)]. Briefly, 0.25 μl of 10 μM miRNA specific forward and 0.25 μl of 10 μM miRNA specific reverse primer, 5 μl of 2× LightCycler 480 SYBRGreen I Mastermix (Roche) and 3.5 μl of RNAse free H2O and 1 μl of cDNA of each samples was combined in 10 μl reaction and subjected to qPCR performed on LightCycler 480 (Roche).
[10 (link)]. Briefly, single tube poly (A) synthesis and reverse transcription was performed with 100 ng of RNA in a final volume of 10 μl combined with 1 μl MuLV reverse transcriptase (New England Biolabs), 1 μl of poly (A) polymerase (New England Biolabs), 1 μl 10x poly (A) polymerase buffer, 1 μl 0.1 mM ATP, 1 μl 10 μM RT-primer (5′-CAGGTCCAGTTTTTTTTTTTTTTTVN where V is A, C and G and N is A, C, G and T). The reaction was incubated at 42°C for 1 hour followed by enzyme inactivation at 95°C for 5 minutes. A panel of three technical cDNA replicates for each of the three biological samples per condition was constructed. The cDNA was diluted 5× before used in qPCR reactions.
miR-specific qPCR was performed as previously described in
[16 (link)]. Briefly, 0.25 μl of 10 μM miRNA specific forward and 0.25 μl of 10 μM miRNA specific reverse primer, 5 μl of 2× LightCycler 480 SYBRGreen I Mastermix (Roche) and 3.5 μl of RNAse free H2O and 1 μl of cDNA of each samples was combined in 10 μl reaction and subjected to qPCR performed on LightCycler 480 (Roche).
Anabolism
angiogenin
Biopharmaceuticals
Buffers
Cationic Protein, Eosinophil
DNA, Complementary
Enzymes
MicroRNAs
Murine Leukemia Virus
Oligonucleotide Primers
Poly A
Polynucleotide Adenylyltransferase
Reverse Transcription
RNA-Directed DNA Polymerase
Most recents protocols related to «Cationic Protein, Eosinophil»
CTC-rich blood samples were centrifuged at 300g for 10 min and concentrated to 70 μL. The cell staining was performed with the addition of the following markers and antibodies for 1 h: CellTracker Orange (CTO; Thermo Fisher Scientific C34551), Calcein AM (Thermo Fisher Scientific L3224), PTPRC antibody conjugated with Alexa Fluor 647 (BioLegend 304020), and CD31 conjugated with Alexa Fluor 647 (BioLegend 303111). To improve the viability and RNA quality of the cells, 15 μL of RPMI with 10% FBS (Gibco) and 3 μL of RNase inhibitor (Thermo Fisher Scientific N8080119) were also added. After incubation, 13 mL of PBS was added to dilute the staining reagents. The sample was spun down at 300g for 10 min and concentrated to 45 μL. To achieve optimal buoyancy in an integrated fluidic circuit (IFC), 45 μL of CTCs was mixed with 30 μL cell suspension reagent (Fluidigm 101-0434) to achieve 75 μL of cell mix.
Alexa Fluor 647
Antibodies
BLOOD
Cationic Protein, Eosinophil
Cells
fluorexon
Immunoglobulins
PTPRC protein, human
cDNA was synthesized from RNA extracted from human lung fibroblasts using the iScript cDNA Synthesis Kit (Cat# 1708891, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. We used 1 μg of RNA per 20 μL cDNA reaction. cDNA was synthesized on C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following protocol: A. Priming stage for 5 min at 25 °C. B. Reverse Transcription stage for 20 min at 46 °C. C. RT inactivation stage for 1 min at 95 °C. D. Hold at 4 °C.
The following reaction mixes were used for qPCR: A. 5 μL of TaqMan gene expression master mix (Cat# 4369016, ThermoFisher Scientific, Waltham, MA, USA) B. 3.5 μL of UltraPure DNase/RNase-Free Distilled Water (Cat# 10977015, ThermoFisher Scientific, Waltham, MA, USA) C. 0.5 μL of GAPDH and B2M for duplexed housekeeping gene controls or D. 0.5 μL target FAM primer. One μL of cDNA was added to 9 μL of the aforementioned primer reaction mix. The reaction was performed on the StepOne Plus Real-time PCR machine by Applied Biosystems (ThermoFisher Scientific, Waltham, MA, USA) using the following protocol: Holding stage (1) 15 min at 48 °C, (2) 10 min at 95 °C. Cycling Stage (1) 1 min at 95 °C, (2) 1 min at 60 °C for a total of 40 cycles. The target gene 2−ΔCT values normalized to GAPDH or B2M were graphed and statistically analyzed in GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA, USA). All qPCR primers utilized are listed inTable S5 .
The following reaction mixes were used for qPCR: A. 5 μL of TaqMan gene expression master mix (Cat# 4369016, ThermoFisher Scientific, Waltham, MA, USA) B. 3.5 μL of UltraPure DNase/RNase-Free Distilled Water (Cat# 10977015, ThermoFisher Scientific, Waltham, MA, USA) C. 0.5 μL of GAPDH and B2M for duplexed housekeeping gene controls or D. 0.5 μL target FAM primer. One μL of cDNA was added to 9 μL of the aforementioned primer reaction mix. The reaction was performed on the StepOne Plus Real-time PCR machine by Applied Biosystems (ThermoFisher Scientific, Waltham, MA, USA) using the following protocol: Holding stage (1) 15 min at 48 °C, (2) 10 min at 95 °C. Cycling Stage (1) 1 min at 95 °C, (2) 1 min at 60 °C for a total of 40 cycles. The target gene 2−ΔCT values normalized to GAPDH or B2M were graphed and statistically analyzed in GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA, USA). All qPCR primers utilized are listed in
Anabolism
angiogenin
Cationic Protein, Eosinophil
Deoxyribonuclease I
DNA, Complementary
Fibroblasts
GAPDH protein, human
Gene Expression
Genes
Genes, Housekeeping
Homo sapiens
Lung
Oligonucleotide Primers
prisma
Real-Time Polymerase Chain Reaction
Reverse Transcription
Touch
To evaluate the antiviral effect of peptide 110766 in PEDV-infected vero cells, PEDV at MOI 0.01 was first incubated with increasing concentrations of the peptides (3.125, 6.25, 12.5, 25, 50, 100 and 200 μM) for 1 h at 37 °C and then inoculated onto the cells. The inoculums were replaced with fresh DMEM containing 2% FBS and incubated for 12 h. Total RNAs were extracted from the cells in a 24-well plate by using TransZol Up according to the manufacturer’s instructions. Reverse transcription was performed as previously described [25 (link)] using 10 μL of the reaction mixture containing 4.5 μL of the total RNA extraction solution, 2 μL 5×PrimeScript RT Master Mix (TaKaRa, Japan), 3.5 μL RNase-Free Water after 20 min kept under 37 °C, 5 s kept under 85 °C, 16 °C storage. The resulting cDNA was finally stored at −20 °C. The cDNA of all samples was performed by the absolute quantification real-time PCR method, as previously described, namely, using 20 μL of the reaction mixture containing 2 μL cDNA, 0.4 μL upstream and downstream primers, respectively, 10 μL PerfectStart II Probe qPCR SuperMix UDG, 0.4 μL Probe, 6.4 μL nuclease-free water, 0.4 μL RoxReference Dye I(50×). The amplification was carried out as follows: 94 °C for 5 min, followed by 40 cycles of 94 °C for 5 s, 60 °C for 30 s (signal sampling).
angiogenin
Antiviral Agents
Cationic Protein, Eosinophil
Cells
DNA, Complementary
dye 50
Oligonucleotide Primers
Peptides
Porcine epidemic diarrhea virus
Real-Time Polymerase Chain Reaction
Reverse Transcription
Vero Cells
The expression levels of immune response–related genes, including T cell–related CD antigens, cytokines, cytotoxic granule, and transcription factors of regulatory T cells, were measured by qPCR using the Bio–Rad CFX96 system (Bio–Rad, Hercules, CA, USA). Specific primers for GAPDH, CD3, CD4, CD8, IFNγ, TNFα, IL–2, IL–4, and IL–10 were described [11 (link),28 (link)]. Freshly isolated total RNA from the OM and submandibular lymph node specimens were converted to cDNA. The PCR consisted of 5 μL of SsoFast EvaGreen Supermix (Bio–Rad), 3.5 μL of RNase/DNase–free water, 0.5 μL of 5 μM of primer mix, and 1 μL of cDNA in a final volume of 10 μL. Cycling conditions were as follows: 30 s at 95 °C followed by 50 cycles of 1 s at 95 °C and 5 s at 60 °C. A melting curve analysis was performed from 70 °C to 90 °C at the end of each program to confirm the homogeneity of the PCR products. All assays were repeated three times, and mean values were used to calculate gene expression levels. Five 10–fold serial dilutions of each standard transcript were used to determine the absolute quantification, specification, and amplification efficiency of each primer set. Standard transcripts were generated by the in vitro transcription of the corresponding PCR product in a plasmid. The nucleotide sequences were confirmed by DNA sequencing using the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). An Agilent DNA 7500 Kit in an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) was used to validate their quality and concentration. GAPDH gene expression was used as an internal control.
angiogenin
Base Sequence
Biological Assay
Cationic Protein, Eosinophil
CD Antigens
Cytokine
Cytoplasmic Granules
Deoxyribonuclease I
DNA, Complementary
GAPDH protein, human
Gene Expression
Genitalia
IL10 protein, human
Interferon Type II
Nodes, Lymph
Oligonucleotide Primers
Plasmids
Reproduction
Response, Immune
Suppressor Factors, Immunologic
T-Lymphocyte
Technique, Dilution
Transcription, Genetic
Tumor Necrosis Factor-alpha
Nematodes were collected from NGM-Agar plates using chilled RNase-free water and worms were washed 3 times with RNase-free water to remove any residual bacteria (3 min, 500 × g). Excess water was removed and the pellet frozen at −80°C. TRIzol was added to samples before homogenization using a pellet grinder, following which RNA was extracted using a Direct-Zol RNA MiniPrep kit (Zymo Research, Cambridge Biosciences). RNA concentration was measured using a Thermo Scientific NanoDrop One, and 100 ng of RNA was reverse transcribed into cDNA using a Bio-Rad iScript cDNA synthesis kit. Quantitative PCR was performed using Bio-Rad Universal SYBR green Supermix in a Bio-Rad CFX Connect Real-Time System. Relative transcript expression for each gene was determined using the delta-delta Ct method as described previously [2 (link),21 (link)].
Three biological replicates were performed per sample. Significance was determined using Student’s t test or one-way ANOVA with a cutoff of p < 0.05.
Three biological replicates were performed per sample. Significance was determined using Student’s t test or one-way ANOVA with a cutoff of p < 0.05.
Agar
Anabolism
Bacteria
Biopharmaceuticals
Cationic Protein, Eosinophil
DNA, Complementary
Endoribonucleases
Freezing
Gene Expression
Helminths
Nematoda
neuro-oncological ventral antigen 2, human
Student
SYBR Green I
trizol
Top products related to «Cationic Protein, Eosinophil»
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TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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RNase A is a laboratory enzyme that catalyzes the hydrolysis of single-stranded RNA molecules. It is commonly used in molecular biology applications to degrade RNA during DNA purification and other experimental procedures.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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The MiniOpticon CFX 48 real-time PCR Detection System is a compact and versatile thermal cycler designed for real-time PCR analysis. It features a 48-well sample block, allowing for efficient and precise DNA quantification and gene expression studies. The system utilizes fluorescent dye detection technology to monitor the amplification of target sequences in real-time, providing researchers with valuable data during the PCR process.
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The MiRNeasy Mini Kit is a laboratory instrument designed for the extraction and purification of microRNA (miRNA) and other small RNA molecules from a variety of sample types, including cells, tissues, and body fluids. The kit utilizes a silica-membrane-based technology to facilitate the efficient isolation of high-quality miRNA and small RNA.
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SYBR Premix Ex Taq II is a ready-to-use PCR master mix containing SYBR Green I dye, Taq DNA polymerase, and other necessary reagents for real-time PCR amplification.
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RNase A is a ribonuclease enzyme used in molecular biology laboratories. It functions by catalyzing the hydrolysis of single-stranded RNA, cleaving the phosphodiester bonds between nucleotides.