FBS Exosome Depletion Kits

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The Heat&Run® gDNA removal kit removes contaminating gDNA from RNA prior to RT-qPCR.

The kit is based on the recombinant heat labile dsDNase, which is irreversibly inactivated at low temperatures (5 minutes at 58ºC or 15 minutes at 55ºC).

Reverse Transcription can be performed in the same tube, thereby minimizing pipetting steps and reducing hands-on time.

Protocol

The kit contains HL-dsDNase which efficiently removes gDNA from RNA preps before running RT-qPCR.

• gDNA is removed, leaving RNA ready for reverse transcription in the same tube (Figure 1).
• Heat-labile dsDNase can easily be inactivated.
• This procedure minimizes pipetting steps and reduces hands-on time.
• The Heat&Run kit is especially well suited for high throughput experiments.

The high activity of the HL-dsDNase at low temperature and its heat-lability makes it ideal to use in a combination with a heat activated RT enzyme. The contaminating genomic DNA is cleaved at ambient temperature and increasing the temperature over 50°C will inactivate the DNase and start the Reverse Transcriptase.

Do you require gDNA removal in applications other than RT-qPCR? Contact our support team for assistance in implementing dsDNase treatment in your workflow.

Kit Contents

• 10X reaction Buffer
• HL-dsDNase (50 or 250 reactions)

The Heat&Run® gDNA removal kit removes contaminating gDNA from RNA prior to RT-qPCR.

The kit is based on the recombinant heat labile dsDNase, which is irreversibly inactivated at low temperatures (5 minutes at 58ºC or 15 minutes at 55ºC).

Reverse Transcription can be performed in the same tube, thereby minimizing pipetting steps and reducing hands-on time.

What is Apoptosis?

Apoptosis is an essentially normal physiological process that removes now redundant, cells, particularly during embryonic development and early growth. In adult animals the process removes cells that are irreparable. The apoptotic process is also involved in many major diseases such as cancer, where transformed tumour cells have their apoptotic process disabled, permitting cell cycling to continue unchecked. In contrast some forms of senile dementia may result from excessive apoptotic induction of neural cells.

The apoptotic process in mammalian cells is a rapid event (2‐4 hours). Within this short time span an apparently viable cell can be quietly dismantled, to disappear leaving no visible trace of its former existence.

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How is apoptosis detected or measured?

An apoptosis cascade of activators, effectors and regulators has been identified. This in turn led to a range of apoptosis assays being devised to detect and monitor these events. Some laboratories will employ two distinct assays, one selected to detect early (initiation) apoptotic events, while a second assay will target a later (execution) event. Apoptosis assays, based on methodology, can be classified into four major inter‐linked groups:

[1] DNA fragmentation (electrophoresis and nick end labelling, TUNEL).

‍[2] Apoptotic proteases (fluorescently labelled antibodies to the caspases).

[3] Flow cytometric analysis (FACS, incorporating other group assays).

‍[4] Membrane alterations (phosphatidylserine flip).

Biocolor’s APOPercentage assay is based on the latter. Further information can be found under the ‘Mode of Action’ Tab.

How does APOPercentage detect apoptosis?

The mammalian cell membrane has been described as a semi‐fluid mosaic structure, composed of phospholipids with a diverse group of inserted proteins and some cholesterol. The phospholipids are the major components of the membrane and are arranged in the form of a ‘bi‐layer’; which is asymmetric in composition, structure, and function.  

To ensure normal transmembrane functions the phospholipids must be maintained in an asymmetric composition. The process is regulated by ‘flippases’, which catalyse the active transport of aminophospholipids from the outer to inner monolayer. However, in cells undergoing apoptosis, flippase is overwhelmed by the action of another enzyme, termed ‘floppase’ or ‘scramblase’. The net effect is a scrambling of the phospholipid distribution between the inner and outer monolayers.

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The APOPercentage assay utilises an intense, pink-coloured dye reagent which is taken up during in-vitro culture by apoptosis-committed cells. This uptake occurs at the stage of Phosphatidylserine transmembrane movement, as produced by the flipflop mechanism. Dye uptake continues until blebbing occurs. No further dye can then enter the now defunct cell and the dye that has accumulated within the cell is not released (unlike necrotic cells which release dye).  

Since the dye reagent is excluded or not retained by healthy or necrotic cells it therefore acts as a specific label for apoptotic cells.

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How are APOPercentage-labelled cells quantified?

Labelled apoptosis cells may then by conveniently analysed by the following methods:

Direct Analysis
The intense pink colour of the labelled cells can be visually assessed using brightfield microscopy. Apoptosis in substrate-adherent cell populations is therefore readily quantified using image analysis techniques. This technique is the most sensitive with the ability of detecting one single apoptotic cell per well.

Colorimetry protocol
Dye that accumulates within apoptotic cells is released into solution via addition of Dye Release Reagent. The concentration of this intracellular dye is then measured at 550nm using a microplate colorimeter/spectrophotometer.

NB: The APOPercentage assay kit does NOT require the use of a Flow Cytometer.

Limit of Detection

A single cell (via image analysis method)

Detection Method

Colorimetric (550nm) (Endpoint) or Image Analysis based

Measurements per kit

Sufficient for 4×24 well plates or 6×96 well plates

Suitable Samples

Adherent mammalian cells (in-vitro)

APOPercentage kit contents:

1. APOPercentage Dye (1x5ml)

2. Dye Release Reagent (1x150ml)

3. Phosphate Buffered Saline (PBS) (1x120ml)

4. 24-well starter plate.

5. Assay kit manual.

The Colorimetric Protocol requires a Microplate Colorimeter / Spectrophotometer.

Additional 96-well plates will be required for use when reading dye absorbance values.

The Direct Detection Protocol Requires an inverted stage microscope with an attached digital camera.

‍NB: Additional reagents (typically culture medium and suitable apoptosis treatments) may be required for sample preparation prior to assay. Consult manual or contact us for further details.

The APOPercentage™ Apoptosis kit is a dye-based, colorimetric assay for detection and measurement of apoptosis (programmed cell death) during in-vitro cell culture.

Introduction

Magen’s HiPure columns are prepared by high quality glass fiber filter membrane as raw materials through membrane cutting, membrane release, ring release, ring pressing, gland, weighing and other processes. HiPure nucleic acid adsorption columns have the characteristics of long-term stability and high binding capacity. Experiments show that the highest binding capacity and binding efficiency of HiPure nucleic acid adsorption columns are basically unchanged when stored at room temperature for 4 years.

The series of nucleic acid columns produced by Magen Biotech are based on carefully selected imported glass fiber membranes (GF/B, GF/D, GF/F). Columns production processes such as polypropylene injection molding materials, injection molding process, and downstream membrane packing and compression rings are strictly controlled. This is to ensure that the column has extremely high adsorption capacity and long-term stability. Compared with conventional products on the market, Magen’s columns are with varieties, and binding rate will not change when stored at room temperature for 4 years.

C13114 HiPure cfDNA Mini Column Set I (Centrifuge Method)

C13115 HiPure cfDNA Column Set Ⅱ (Vacuum Method)

Details

Specifications

Adsorption Mechanism

Based on the negatively charged DNA skeleton, it has a high affinity for positively charged glass fibers. In high salt and ethanol solutions, DNA/RNA binds to glass fiber and interacts with hydrophilic matrix on silica through hydrogen bond. DNA/RNA is tightly bound. All pollutants can be removed by washing solution. At high salt concentration, nucleic acids selectively bind to silica gel membrane, while other pollutants, mainly proteins, are removed by membrane washing.

Ordering information

Purchase Guide

Note: GF/B pore size is for 1.0μM glass fiber membrane; GF/F pore size is for 0.7μm glass fiber membrane.

Magen’s HiPure columns are prepared by high quality glass fiber filter membrane as raw materials through membrane cutting, membrane release, ring release, ring pressing, gland, weighing and other processes. HiPure nucleic acid adsorption columns have the characteristics of long-term stability and high binding capacity. Experiments show that the highest binding capacity and binding efficiency of HiPure nucleic acid adsorption columns are basically unchanged when stored at room temperature for 4 years.