cell technology


	888 7 ASSAYS (888-727-7297)

Caspase / Apoptosis Detection

	High Throughput Screening - Apo3HTS
	Caspase Poly,1,2,3,7,8,9,10 Detection - FAM-FMK
	Caspase Poly, 1,2,3,8,9,10, Detection - (SR-FMK)
	Antibody Specific Caspase 3 Detection - Apo Active 3 (FITC)
	Antibody Specific Caspase 3 Detection - Apo Active 3 (PE)

NOS/ROS Detection Kits

	Nitric Oxide Synthase Detection Kit - NOS *New!!!*
	Hydroxyl (OH)/ Peroxynitrite (ONOO-)Detection Kit - Fluoro Hydroxyl / Peroxynitrite New!!!
	Hypochlorite (Ocl-) Detection Kit - Fluoro Hypochlorite New!!!

Fluorescent Enzymatic Assays

	Catalase Detection Kit - Fluoro Catalase
	Monoamine oxidase A&B detection - Fluoro MAO
	Semicarbazide sensitive amine oxidase detection - Fluoro SSAO
	Hydrogen Peroxide detection - Fluoro H₂0₂

Mitochondria Membrane Potential detection

	Mitochondria Membrane Potential - JC 1
	MM Potential detection for Flow Cytometry - Mito Flo

aCella - Bioluminescence Assays Kits

	Non Radioactive Assay for Cytotoxicity - aCella TOX
	Monitoring Acetylcholinesterase activity - aCella AChE New!!!

Cytotoxicity

Antibody Mediated Cytotoxicity - ACT 1

ELISA Products

	Conjugate Diluents
	Assay Diluents
	Sample Diluents
	Wash Buffers
	Plate Coating Buffers
	Block Buffers

Cathepsin B,K,L Detection Kits

	Substrate based Assay to detect protease activity in hole living cells

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

References:

Ken-ichi Setsukinai, Yasuteru Urano, Katsuko Kakinuma, Hideyuki J. Majima , and Tetsuo Nagano. Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 5, Issue of January 31, pp. 3170–3175, 2003
Manufactured: by Daiichi Pure Chemicals Co. Ltd. Japan

The following kits are available:
Catalog #	Contents	Size*	Price
FLOCL 100-2	APF & HPF 1 vial each	150 Tests *	$345
Not for sale in Japan *Test size will depend on dilution and volume used per test. For example:

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

* Please call 888 7 ASSAYS (888-727-7297) or email info@celltechnology.com for volume pricing

Product Data Sheet

Protocol

MSDS

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.