Recipe. Add 1ml of % Diethylpyrocarbonate (DEPC) to ml distilled water. Mix well and let set at room temperature for 1 hour. Autoclave. Let cool to room temperature prior to use. It is commonly used at a concentration of % to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add ml DEPC to ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37°C.
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Peer Reviewed Literature. Product Usage Information. Global Support. Medical Affairs. Local Sales Support. Your Cart. Current Items 0. Buffers often are overlooked and taken for granted by laboratory scientists until the day comes when a bizarre artifact is observed and its origin is traced to a bad buffer.
Although mistakes in the composition of buffers have led occasionally discoveries such as the correct number of human chromosomes Arduengo,using the proper buffer, correctly prepared, can be key hwo success in the laboratory. Most simply, a buffer functions to resist changes in hydrogen ion concentration as a de;c of internal and environmental factors.
However, biologists often think of buffers as doing much more: providing essential cofactors for enzymatically driven reactions, critical salts, and even essential nutrients for cells and tissues. However, when the basic function of a buffer system, resisting changes in hydrogen ion concentration, is overlooked, experimental artifacts and other problems mmake follow.
Here we examine the basic chemistry of buffer systems and how that chemistry applies eater reactions in experimental biological systems. Buffers consist of a weak acid HA and its conjugate base A — or dater weak base and its conjugate acid.
Weak acids and bases do not completely dissociate treatee water, and instead exist in solution as how to power a breadboard equilibrium of dissociated and undissociated species. Consider acetic acid. In solution acetate ions, hydrogen ions and undissociated acetic acid exist in equilibrium. In this way, pH is maintained as the three species constantly adjust to restore equilibrium. All buffers have an optimal pH range over which they are able to moderate changes in hydrogen ion concentration.
This range is a factor what is 270 percent of 60 the dissociation constant of the acid of the buffer What causes chalky white feces a and is generally defined as the pK a —logK a value plus or minus one pH unit.
InNorman Good and colleagues set out to define the best buffers for biochemical systems Good et jake. Good set forth several criteria for such buffers:. Good et al. No matter what buffer you choose, you need to consider effects of temperature and environment on the buffer and ensure that the buffer is compatible with your system.
As discussed previously factors like temperature and concentration can greatly influence the pK aand therefore, the pH mqke over which a buffer system is most effective. Careful preparation of buffers is important for successful and reproducible experiments. Because changes in temperature can be associated with a shift in dissociation, prepare your buffers at the temperature at which you will be performing your experiments. If your experiment involves a change in temperature, choose how to make depc treated water buffer cepc a pK a that accommodates it.
Changes in concentration also can be associated with a shift in dissociation, so if you plan to maintain buffer stock solutions, make sure that the pH adjustment is made after you have diluted the stock to the desired concentration and equilibrated it at the appropriate temperature.
Or, at the very least, check the pH after dilution. Many buffer materials are watrr as crystalline acids or bases e. When these materials are dissolved in water, the pH of the solution is not near the pK aand the pH must be adjusted using the appropriate acid or base before the solution will become a suitable buffer.
If the crystalline buffer material is an acid, then pH can be adjusted to the desired pH with a base that will not add mke unwanted counter ion. If the material is a base, then an appropriate acid may be used. Water can be added to reach the final desired volume after the desired pH is obtained.
Deepc buffers, however, are not made by dissolving a crystalline acid or base then adjusting the pH to bring the solution close to the pK a. Instead the buffer system is prepared by mixing two components, such as the free acid or base and the salt, in jow ratios to achieve the desired pH. For instance, a 0. Sodium citrate buffer solutions hoa be made and adjusted to the desired pH by mixing citric acid and trisodium citrate. Other buffers are made by mixing the buffer component and its conjugate acid or base using Henderson-Hasselbalch calculations.
For instance, phosphate buffers are made by mixing monobasic and dibasic sodium phosphate solutions in a specific ratio. Freated bicarbonate what film has won the most oscars systems are made by mixing solutions of sodium carbonate and ho bicarbonate.
The use of pH meters seems almost intuitive; however, pH meters must be maintained properly and electrodes cleaned and filled, and pH calibration buffers need to mak correctly prepared and free of how to make depc treated water. When using a pH meter, temperature is important because the pH meter electrode is temperature-dependent.
The meter should be set to ambient temperature while pH is being measured. Unfortunately, the pH meter is often the most neglected piece of equipment in the laboratory. If the pH meter is being abused, the treatee of common laboratory buffers may be incorrect, and the downstream consequences could be disastrous. Using acetic acid as an example, the equilibrium relationship of a weak acid, hydrogen ion and the conjugate base can be expressed mathematically as:. We can rearrange that equation to express hydrogen ion concentration hwo terms of the equilibrium constant and the watrr acetic acid and acetate ion.
Using this equation, you can calculate pH when concentrations of acid and base and pK a are known. The pK a terated a buffer system determines the pH range at which that buffer is most effective.
To create ml of a 0. Solution B: 0. Solution A: 0. Note: The dibasic stock sodium phosphate may be somewhat harder to dissolve; adding a little heat may help. For 2 liters of buffer, add Add 20ml of DEPC-treated 0. Bring the final volume to 2 liters with DEPC-treated water. Filter sterilize and dispense into aliquots.
What Makes a Good Buffer?
How to Make DEPC-Treated Water and Tris Buffer:Solutions (water and other solutions) should be treated with % DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of % to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. DEPC-Treated Water is suitable for use with RNA. It is prepared by incubating % DEPC and is then autoclaved to remove the DEPC. Although autoclaving does inactivate DEPC by causing hydrolysis of DEPC, EtOH (ethanol) and CO 2 are, however, released as reaction by-products. DEPC-Treatment of Water Introduction Many experimental procedures in biological research require the use of RNase-free water to prevent the degradation of RNA. RNase-free water is generated when distilled water is treated with diethylpryrocarbonate (DEPC), .
The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miRa-5p during Leishmania donovani infection. We found involvement of miRa-5p in phagocytosis and survivability of parasites.
Moreover, miRa-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Additionally, we explored the novel role of super enhancer SE , a cis-acting regulatory component, to enrich miRa-5p expression during infection. Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miRa-5p gene during infection.
Visceral leishmaniasis VL , caused by protozoan parasites Leishmania donovani , is the most severe form of leishmaniasis and is highly lethal if left untreated. Major obstacle for successful therapy of VL originates from the life-long immune-suppression triggered in the post kala-azar dermal leishmaniasis PKDL patients during infection. Identification of molecular principles behind such immune-suppression will add success in VL therapeutics.
Herein, we explored the indispensable role of miRNAa-5p in conversion of M1 to M2 type during infection. Both in vitro and in vivo miRNA silencing established miRa-5p as an imperative negative regulator ofM1 polarization. Computational analysis as well as immune precipitation based experiments authenticated that L.
In short, our current findings established a previously unrecognized role of BRD4-depdendent super enhancers in orchestrating persistent transcription of macrophage miRa-5p which in turn promotes M2 polarization during L.
PLoS Pathog 17 2 : e This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Visceral leishmaniasis VL , caused by Leishmania donovani , results in almost 0.
A major hurdle in the successful treatment of the infection is the induction of chronic immune suppression in patients [ 3 ]. Recent studies indicated that the final outcome of VL largely depends upon dynamic interchanges between classically activated M1 macrophage, which has leishmanicidal properties, and alternatively activated M2 macrophage which supports parasite survival [ 5 , 6 ]. These transcription factors in turn mediate activation of induced nitric oxide synthase i NOS in M1 macrophages.
Muxel et al. We have currently undertaken differential expression analysis of immune-regulating miRNAs from L. Upon obtaining significant enrichment of miRa-5p, we further testified involvement of the same in stimulating M2 polarized macrophage during L. We found that, L. During endotoxic stimulations, persistent transcription of miRa has been previously addressed which signified a spectacular transcriptional regulatory element formation, at upstream cis-acting regions of miRNAs-encoding gene called super-enhancers SEs [ 23 — 24 ].
Therefore, we explored the involvement of BRD4 and p and investigated their nuclear retention and chromosomal accumulation upon enhancer domain of miRa-5p gene. Virulence of the parasite strains was maintained by passaging through 4—6 week old hamsters, which were also bred and maintained in the animal care facility of CSIR-IICB. Metacyclic promastigotes, enriched in ficoll gradient [ 26 , 27 ] were used throughout the study to infect BMDMs and animals.
For infection synchronization, extracellular parasites were washed with warm 20 mM phosphate buffered saline PBS and macrophages were incubated for the indicated times for infectivity related experiments. Infection related studies were performed 24 hrs post transfection. LDU was represented by the number of amastigotes calculated in nearly macrophages from each slide of organ impressions multiplied by the respective organ weight in milligram mg.
Parasite burden was expressed as fold logarithm scale of the highest dilution containing viable parasites, and the mean values of three mice per group were represented.
To determine the rate of phagocytosis, 5 X 10 7 L. Phagocytosis of zymosan particle by uninfected BMDM was used as positive control for the assay. After diluting the cDNA 25 times, 1. All the experiments were performed in triplicates. Ct values for control and test genes were analyzed using Light cycler 96 software and fold change was calculated using the following formula:. Details of primers are provided in S1 Table.
Membranes were then washed three times with wash buffer TBS containing 0. Densitometry data was analyzed using Image lab software version 5. The uncropped blots are provided in S2 Fig and the details of the antibodies are provided below-. After washing the excess fixative, cells were permeabilized using 0. Excess secondary antibodies were washed with PBS vigorously and nuclei were stained with Hoescht Molecular probes, Thermo scientific, USA for 10 mins in the dark.
Experiments were performed in triplicates and data were analyzed using graph pad prism version 5. Experiments were done in triplicate and during acquisition of signal, gating of macrophage population was performed using auto polygon gating Using BD FACSDiva software on the basis of unstained compensated population.
Then after doublet discrimination, each population was further analyzed for cytokine expressions. The final populations were represented as histogram plots denoting mean fluorescence intensity MFI for each cytokine. The denaturant was quenched by adding mM glycine and incubated for 5 mins followed by rinsing and scraping the cells with cold PBS. The primers used for the amplification of bp segment of miRa-5p gene enhancer sequence, are mentioned in S1 Table.
The PCR product was resolved on 1. The supernatant used was the cytosolic fraction and the pellet was further homogenized in the presence of nuclear buffer B 5 mM HEPES, 1. The supernatant was collected as the nuclear fraction and the protein content was measured by Bradford assay before western blotting. Nitric oxide generated in culture supernatant of different groups of BMDMs was measured by Griess assay. Absorbance at nm was then measured.
The graph for nitric oxide was generated using graph pad prism version 5. All the experiments were performed in biological and experimental triplicates. From this screening, miRa-5p was selected for further mechanistic evaluation of its involvement in infection-induced M2 polarization. H 24 hrs post infection, survival rate of unstained parasites was measured by geimsa staining and represented as number of amastigotes per BMDMs.
Each experiment has been performed in triplicates. RT-PCR experiment based analysis of mature miRa-5p titer suggested that miRa-5p level enhanced in positive correlation with doses and times of parasite infections Fig 1B and 1C. To correlate in vitro findings with in vivo infection, miRa-5p expressions were also evaluated in spleen and liver of 15, 30, 45 and 60 days infected animals, which showed a successive enrichment pattern of the miRNA with time of infection Fig 1D.
These findings cumulatively indicated possible involvement of miRa-5p during L. In order to understand the significance of miRa-5p for parasite entry and survival inside BMDMs, macrophages were transfected with anti-NC and anti- miRa-5p inhibitor oligos. Percentage of CFSE signal reflects the amount of parasite phagocytized.
However phagocytic capacity of healthy macrophages were assessed uptake of pHrodo green zymosan particles during the assay, which showed significant uptake of the particles. These data cumulatively suggested that miRa-5p might play a vital role in parasite phagocytosis in BMDMs.
Similarly another set of BMDMs were infected with unstained parasites for 24 hrs. Geimsa staining based parasite burden calculation corroborated with the flow cytometry data. These findings suggested plausible involvement of miRa-5p dependent survival of parasites inside macrophages.
D Expression of M2 polarization markers were analyzed in L. Expression of transcription factors essential for directing either M1 E-H or M2 I-K polarization of macrophages, were determined in anti-NC or antia transfected and L. These finding indicated a positive relationship between miRa-5p and M2 plasticity of macrophages. Next differential expression profiling of M2 polarization markers from anti-NC or anti-miRa-5p-treated and L.
Besides surface markers, expression profiling of some transcription factors has been undertaken to elucidate global regulation of miRa-5p upon M2 polarization. M1 polarized macrophages are characterized by low Arginase 1 Arg 1 , high induced nitric oxide synthase i NOS and enhanced nitric oxide levels. Polarity of macrophages upon pathogenic challenge largely depends upon its cytokine profile. Upon entry into the macrophages, L. Cumulatively, we found that miRa-5p is crucial for enrichment of M2 cytokines during infection.
Mean fluorescence intensity MFI of each cytokine was further represented using bar diagrams. Therefore we evaluated the effect of parasite infection upon nuclear translocation of p-p65 by both confocal microscopy and western blotting. Co-localization co-efficient values and percent overlap of pp65 signals with nuclear Hoescht signals suggested positive nuclear accumulation of phospho-p65 subunit ser in miRa-5p inhibited and L.
Interestingly, parasites were unable to suppress phosphorylation as well as nuclear translocation of p65 subunit upon deletion of miRa-5p which indicated the microRNA as a major nexus for L.
B, C Co-localization coefficients of p-p65 alexa Fluor AF signals with Hoescht signals were determined while capturing the images at 63X oil immersion objective with 2. Post hoc analysis was performed using Leica Application Suit X LAS-X software and co-localization coefficients with percent overlap of the signals were plotted for each condition. D Nuclear and cytosol fractions of the BMDMs were isolated and expression of p-p65 was analyzed by western blotting. Our next question was how the infection mediates sustained and prolonged expression of mature miRa-5p RNA.
Super enhancer SE elements are outstanding cis-acting elements, which transcriptionally control sustained RNA expression under different stimulations.
We therefore experimentally validated formation of SE complexes during L. Immunoblotting of whole cell lysate and nuclear fractions suggested significant upregulation and nuclear translocation of BRD4 and p during L. Besides that, confocal microscopy based validation of nuclear retention of BRD4 as well as RpbI also substantiated the western blot data Fig 6B.
Moreover immune-precipitation studies showed a strong interaction between these three proteins Fig 6D. Nuclear translocated proteins were normalized with Histone H3. D Western blot of p and RpbI, immune-precipitated by anti-BRD4 antibody and western blot of BRD4, immuno-precipitated by anti-p antibody using co-immuno precipitation assay. K Schematic diagram of chemical cross-linking based chromatin immune-precipitation of super enhancer region of miRa-5p gene using anti-BRD4 antibody.
Percent input represents normalized amount of enhancer DNA enriched against total input. Taken together, these data affirmed SE complex driven transcription of miRa-5p during L.
Before injecting inhibitor oligos, infection burden in two animal groups were analyzed by Leishman donovan unit LDU counting and Limiting dilution assay LDA of spleen and liver.
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