CELL TECHNOLOGY Fluorescent Hypochlorite Detection Kit

Okwu mmalite

The two new novel probes, Aminophenyl fluorescein (APF) and Hydroxyphenyl fluorescein (HPF) developed by Tetsuo Nagano et. al., are selective dyes for the detection of highly reactive oxygen species (hROS). The compounds themselves are not very fluorescent, however, when reacted with hROS (hydroxyl radical: .OH, Peroxynitrite: ONOO-, and hypochlorite: -OCl), APF and HPF exhibit strong dose-dependent fluorescence. Furthermore, using these probes together, hypochlorite (-OCl) can be selectively detected from hydroxyl radical: (.OH) and Peroxynitrite: (ONOO-). HPF/APF can be used to differentiate hROS from H₂O₂, nitric oxide (NO), and superoxide.

Ụkpụrụ nyocha
The principle of the assay is based on the fluorescence change upon reaction with hROS. The diagram shows the chemical structure transformation leading to fluorescence.

Nchekwa
Aminophenyl fluorescein (APF) and Hydroxyphenyl fluorescein (HPF) should be stored at 4°C. Protect from light until ready to use. The diluted material must be used immediately and discard any unused diluted material.

Ịdọ aka ná ntị na ịkpachara anya

• For Research use only. Not for use in diagnostic procedures.
• Practice safe laboratory procedures by wearing protective clothing and eyewear.

Ọdịnaya ngwa

1. Kit: Hydroxyl radical/Peroxynitrite Detection: Part# 5020 (Cat # FLHPF100-2)
   1. 1 vial: Part# 4012 HPF 5mM solution in DMF
2. Kit: hROS Detection: Part# 5027 (Cat # FLAPF100-2)
   1. 1 vial: Part# 4011 APF 5mM solution in DMF
3. Kit: Hypochlorite Detection: Part# 5021 (Cat # FLOCL100-2)
   1. 1 vial: Part# 4011 APF 5mM solution in DMF
   2. 1 vial: Part# 4012 HPF 5mM solution in DMF

Materials and Equipment Required but not Supplied

• Fluorescence plate reader / Fluorescent Microscope
• Serum/BSA Free buffer

Preparation of Reagent Working Solutions

Cells should be loaded with HPF and APF in serum/BSA free media. HPF and APF can be diluted to a 10μM loading solution in modified HBSS with 10mM Hepes, 1.0mM MgCl₂, 2.0mM CaCl₂, and 2.7mM glucose or Krebs-Ringers phosphate buffer (with 114mM NaCl, 4.6mM KCl, 2.4mM MgSO₄, 1.0mM CaCl₂, 15mM NaH₂PO₄NaHPO₄, pH 7.4).

Nlebara anya: As Phenol red may interfere with the assay, care should be taken to avoid using media containing Phenol red.

Table of Reactivity

ROS	HPF
Hydroxyl Radical: .OH	730
Peroxynitrite: ONOO-	120
Hypochlorite: -OCl	6
Oxygen Radical: ¹O₂	5
Superoxide: O₂⁻	8
Hydrogen Peroxide: H₂O₂	2
Nitric Oxide: NO	6
Alkylperoxyl Radical: ROO.	17

Assay Protocol: Cells

1. Rinse cells with modified HBSS or Krebs-Ringers phosphate buffer (as described above step V 1.).
   See Technical note 1 below.
2. After the final wash, adjust cells to desired concentration (.1 to 1 XIO cells/mL) in HBSS or
   Krebs-Ringers phosphate buffer. Adherent cultures do not need detachment before loading the dye.
   Note: The optimal cell concentration may vary, and should be empirically determined by each investigator.
3. Loading the cells with APF / HPF. The final concentration of APF or HPF should be in the range of I-IOHM. For example dilute APF/HPF 1:10 in HBSS or Krebs-Ringers phosphate buffer to yield a 500k1M stock solution. Add 2k1L of the diluted APF / HPF to 100 PL of sample to yield a final concentration of IOVIM.
   See Technical note 2 below.
4. Incubate cell for 30-60 minutes between 25-37 C in the dark.
   Mara: Each investigator should determine the optimal loading temperature and length of incubation for their particular application.
5. Without washing, activate cells according to your experimental protocol.
6. Measure fluorescence using a fluorescence plate reader or Fluorescent Microscope, utilizing excitation: 488nm and emission: 515nm.

Figure 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 (10μM 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 filters 1

Ihe ndetu nka

1. DMSO is a scavenger of Hydroxyl Radical.
2. 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 and no increase in fluorescence in HPF loaded cells.

Ntụaka:

1. 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

Cell Technology, Inc. 25242 Co Rd 95, Davis, CA 95616 www.celltechnology.com 

Ozi mkpọtụrụ

• adreesị: Cell Technology Inc, 25242 Co Rd 95, Davis, CA 95616 USA
• Ekwentị: 800-824-8540
• Ozi izugbe: info@celltechnology.com
• Ahịa: sales@celltechnology.com
• Ajụjụ nka nka: techsupport@celltechnology.com
• Websaịtị: www.celltechnology.com

Ajụjụ a na-ajụkarị

Can the kit be used for diagnostic procedures?

No, the kit is for research purposes only and should not be used for diagnostic procedures.

How should the materials be stored?

APF and HPF should be stored at 4°C and protected from light until ready to use.

Akwụkwọ / akụrụngwa

CELL TECHNOLOGY Fluorescent Hypochlorite Detection Kit [pdf] Akwụkwọ ntuziaka FLOCL100-2, FLHPF100-2, FLAPF100-2, Fluorescent Hypochlorite Detection Kit, Hypochlorite Detection Kit, Detection Kit, Kit

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