Hypochlorite (Ocl-) Detection Kit

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Hypochlorite Detection Kit™
Fluorescent Ocl (Hypochlorite) Detection Kit

Key Benefits:

Quenched Cell permeable dye
Can be used with Cell Lysates, Tissue Homogenates
One Step, No wash Homogenous assay.
Adaptable to High throughput assay platforms
Can monitor multiple time points to follow real time kinetics
Non-destructive cell based assay allows monitoring of additional parameters
Applications - Fluorescence Plate Reader / Flow cytometry / Fluorescent Microscope

Introduction:

The two new novel probes, Aminophenyl fluorescein (APF) and Hydroxyphenyl fluorescein (HPF) developed by Tetsuo Nagano et. al. (1), are selective for the detection of highly reactive oxygen species (hROS). Both probes have little reactivity towards other ROS such as: singlet oxygen (0₂¹), superoxide (O₂^-•), hydrogen peroxide (H₂0₂), nitric oxide (NO^•), and alkyl peroxide (RO₂^•) (see table below)¹. HPF/APF are cell permeable and can be used in combination to detect hypochlorite (^-OCl) production in cells (see fig 1). Hypochlorite can be detected by loading two samples, one with APF and the other with HPF. Hypochlorite production is visualized by increase in fluorescence of APF loaded cells and no increase in fluorescence in HPF loaded cells.

Assay Principle:

Reactivity:

Reactivity Profile of APF/HPF:
ROS (RFU)	HPF (RFU) Ex:499 Em:515	APF (RFU) Ex:499 Em:515	DCFH-DA (RFU) Ex:500 Em:520
Hydroxyl Radical: ^•OH	730	1200	7400
Peroxynitrite: ONOO^-	120	560	6600
Hypochlorite: ^-OCl	6	3600	86
Oxygen Radical: ¹O₂	5	9	26
Superoxide: O₂^-•	8	6	67
Hydrogen Peroxide : H₂0₂	2	1	190
Nitric Oxide: NO	6	1	150
Alkylperoxyl Radical: ROO.	17	2	710
Autoxidation	1	1	2000

The following kits are available:
Catalog	Contents	Size*	Price
FLOCL 100-2	APF & HPF 1 vial each	150 Tests	€390

Dilution of APF/HPF	Volume of test	Numer of tests
1:500 (10mM final)	0.2 mL	147
1: 1000 (5mM final)	0.2mL	294

Fig 1. : Detection of Hypochlorite ( ^-OCl ) in neutrophils. Neutrophils were isolated from porcine blood, washed in Krebs-Ringers phosphate buffer (as described in step V 1. above) and seeded in glass bottom dishes. The cells were then loaded with APF or HPF (10mM final) by incubation for 30 minutes at room temperature. The Dye-loaded neutrophils were stimulated with PMA (2ng/mL). Fluorescence images were acquired before and 10 minutes after stimulation. Excitation: 488nm emission: 505-550 nm barrier filters¹. Hypochlorite production can be detected with increase APF fluorescence and no increase in HPF fluorescence.