Respiratory Syncytial Virus A (RSV-A) TaqMan RT-PCR Detection Kits

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.

Overview

• Detection kits for Cryptosporidium
• CE-IVD marked version available for in vitro diagnostic use
• Available in TaqMan format for analysis

Cryptosporidium is a parasite found in water that causes an infection in mammals termed cryptosporidiosis. It is one of the most common water-borne diseases and is found world-wide. It affects the intestines of mammals and typically causes an acute short-term infection. The most common symptom is self-limiting diarrhea in healthy individuals, however in immunocompromised individuals the symptoms are particularly severe and often fatal. There is no specific treatment for cryptosporidiosis other than fluid rehydration and management of any pain. Therefore early detection of Cryptosporidium in water is the foremost action to prevent the infection.

Cryptosporidium TaqMan RT-PCR Kit, 100 reactions

• Ready to use format, including Master Mix for the target and PCR control to monitor for PCR inhibition and validate the quality
• Specific Primer and Probe mix for the pathogen/virus/viroid of interest
• Primer and Probe mix
• Positive and negative control to confirm the integrity of the kit reagents

Cryptosporidium TaqMan RT-PCR Probe/Primer Set and Controls, 100 reactions

• Specific Primer/Probe mix and Positive Control for the pathogen/virus/viroid of interest
• Nuclease-free water
• Can be used together with Norgen’s RT-PCR Master Mix (#28113) or customer supplied master mix

Details

Supporting Data

Figure 1 / 3

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Storage Conditions and Product Stability
All kit components can be stored for 1 year after the date of production without showing any reduction in performance.

All kit components should be stored at -20°C upon arrival.

Component	Cat. TM39150 (100 preps)	Cat. TM39110 (100 preps)
MDx TaqMan 2X PCR Master Mix	2 x 700 μL	–
Cryptosporidium Primer & Probe Mix	280 μL	280 μL
Cryptosporidium Positive Control	150 μL	150 μL
Nuclease-Free Water (Negative Control)	1.25 mL	1.25 mL
Product Insert	1	1

Opentrons Flex 1-Channel pipettes enable highly accurate pipetting, optimized for automation.

Flex pipettes use air displacement technology to offer highly accurate pipetting with pipette volume ranges from 1 to 50 µL and 5 to 1000 μL. Smart sensors support automatic calibration, real-time positioning, and error detection. Choose the pipettes that fit your workflow — the gantry supports any two Flex 1-Channel or 8-Channel Pipettes — and swap them out yourself when your workflow changes.

Opentrons Flex 1-Channel pipettes enable highly accurate pipetting, optimized for automation.

Flex pipettes use air displacement technology to offer highly accurate pipetting with pipette volume ranges from 1 to 50 µL and 5 to 1000 μL. Smart sensors support automatic calibration, real-time positioning, and error detection. Choose the pipettes that fit your workflow — the gantry supports any two Flex 1-Channel or 8-Channel Pipettes — and swap them out yourself when your workflow changes.