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

• Extract total RNA, including virus & viroid RNA
• Robust lysis buffer is well-suited to even challenging samples such as pine needle, grape leaf, etc
• Isolate total RNA (including microRNA) without phenol
• Isolated RNA is of high quality, integrity and diversity
• Also available in 96-well format for high throughput applications
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

Norgen’s Plant/Fungi Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA, including virus and viroid RNA, from a wide range of plants. Total RNA can be purified from fresh or frozen plant tissues, plant cells or filamentous fungi samples using this kit. All sizes of RNA are purified, including microRNA (miRNA) . The procedure is rapid and convenient. 

The RNA is purified without the use of phenol or chloroform. The purified RNA is of the highest quality, and can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.  

Norgen’s Plant/Fungi Total RNA Purification Kit is also available in a 96-well (High Throughput) format for high throughput applications.  Purification with the 96-well plates can be performed using either a vacuum manifold or centrifugation.

Details

Supporting Data

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Kit Specifications – Spin Column
Maximum Column Binding Capacity	50 μg
Maximum Column Loading Volume	650 μL
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Maximum Amount of Starting Material: Plant Tissues Plant Cells Fungi	50 mg 1 x 106 cells 50 mg (wet weight)
Average Yield* 50 mg Tomato Leaves 50 mg Tobacco Leaves 50 mg Plum Leaves 50 mg Grape Leaves 50 mg Peach Leaves	60 μg 60 μg 32 μg 35 μg 30 μg
Time to Complete 10 Purifications	30 minutes

* Yield will vary depending on the type of sample processed.

* Yield will vary depending on the type of sample processed.

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Select Plants and Fungi Tested with the Norgen Plant/Fungi Total RNA Purification Kits

Plants

Tobacco (Nicotiana tabacum)
Tomato (Lycopersicon esculentum)
Pepper (Capsicum annuum)
Potato (Solanum tuberosum)
Arabidopsis thaliana1
Peach (Prunus persica)
Apple (Malus sp.)
Pear (Pyrus sp.)
Grape vine (Vitis sp.)
Plum (Prunus sp.)
Palm (Arecaceae)
Pine needle (Pinaceae)
Strawberry
Raspberry
Blackberry
Herbs
Persimmon (Ebenaceae)
Potato tuber (Solanum)
Plum fruit
Citrus
Vanilla bean
Cotton (Gossypium)
Mangrove
Chrysanthemum
Grape berry skin
Kiwi leaves
Peach (fruits and flowers)
Soy bean (legume)
Eastern White Red Cedar
Corn leaves
Cucumber leaves

Fungi

Aspergillus niger
Mucor racemosus
Cladosporium cladosporioides
Fusarium oxysporum
Penicillium sp.
Botrytis cinerea (Botryotinia fuckeliana)
Pichia sp.
Rhizopus oryzae
Alternaria tenuissima

Component	Cat. 25800 (50 preps)	Cat. 31350 (100 preps)	Cat. 25850 (250 preps)	Cat. 31900 (192 preps)
Lysis Buffer C	60 mL	1 x 30 mL, 1 x 60 mL	3 x 60 mL	2 x 60 mL
Wash Solution A	38 mL	38 mL	1 x 18 mL, 2 x 38 mL	2 x 38 mL
Elution Solution A	6 mL	6 mL	20 mL	20 mL
Filter Columns	50	100	250	–
Spin Columns	50	100	250	–
96-Well Plate	–	–	–	2
Adhesive Tape	–	–	–	4
Collection Tubes	100	200	500	–
96-Well Collection Plate	–	–	–	2
Elution Tubes (1.7 mL)	50	100	250	–
96-Well Elution Plate	–	–	–	2
Product Insert	1	1	1	1

Technical Parameters

Heat Sealing Time:		0.5~2s		Heat Sealing Tube Outer Diameter:		2~6mm
Indicator Light Colors:		Red/Blue		RF Frequency:		40.68MHz
Grounding Protection:		Class I		Power Supply		100V~120V/220V~240V AC
Power Consumption:		250W during operation,10W in standby		Dimensions:		175×155×315mm
Weight:		5.5kg		Fuse:		3A/250VAC 5*20mm
Operating Temperature:		0~40℃		Storage Temperature:		-20~70℃

A device that uses the principle of high-frequency oscillation to generate heat through the friction of molecules in PVC hoses, thereby melting the PVC hose and achieving the sealing effect. This device is also referred to as a pipe sealing machine in some places.