Global Phospholipid Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence (BN00937)

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BN00937
€589

Description

ELISA Kit Technical ManualMSDS

Global Phospholipid Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence

Phospholipids are major component of the bilayers of all plasma membranes. A single phospholipid molecule consists of a phosphate group on one end, called the "head," and two side-by-side chains of fatty acids that make up the "tails". The phosphate head groups can be modified with organic molecules such as Choline (Cho). Cho-containing phospholipids (Phosphatidylcholines; PC) are critical for structural membrane integrity, cellular metabolism and signaling either as individual molecules or precursors of secondary messengers. Changes in global synthesis of Cho-containing phospholipids are an essential parameter in analysis of cellular response to both, physiological and pathological conditions, environmental stress, or drug treatment. To date, phospholipids biochemistry, cell biology and metabolism remain obscure, due to limited methods for their direct cellular visualization. Assay Genie's Global Phospholipid Synthesis Assay Kit offers a simple and robust method to label and visualize newly synthesized phospholipids in vivo. Based on the metabolic incorporation of the choline analogs directly into their structure, modified phospholipid molecules can be detected with high sensitivity and spatial resolution by click chemistry with azide-containing dyes. This kit enables analyses of global biosynthesis, subcellular localization and turnover of Cho-containing phospholipids in cells. Cells show strong incorporation of Cho analogs into all classes of phospholipids that can be assayed by fluorescence microscopy, or quantified by FACS. We provide sufficient materials for 100 assays

Figure: Analysis of metabolic labeling of phospholipids in proliferating cells. (A) Jurkat cells (1X10 6 cells/ml) were pre-treated with vehicle (black line) or cultured in presence of 1X Phospholipid Label (red line) for 24 hours at 37°C prior to 1 hour incubation with Phospholipase D (blue line). Modified phospholipid molecules were detected according to the kit protocol and red fluorescence was analyzed by FACS in FL-2 channel. Decrease in signal caused by hydrolysis of Cho-containing head groups via Phospholipase D activity confirms that red fluorescence is the result of Phospholipid Label incorporation. (B) BALB/3T3 cells (10 5 cells/ ml) cultured in presence of 1X Phospholipid Label for 24 hours at 37°C and processed according to kit protocol. Choline-containing phospholipids were detected by Fluorescence Microscope. according to kit protocol. Total DNA staining (C) confirms that red fluorescence is the result of Phospholipid Label incorporation.

Key Information Description

Product SKU

BN00937

Size

100 assays

Kit Summary

  • Detection method- Flow Cytometry (Ex/Em 488/(530/590) nm) and Fluorescence Microscopy (Ex/Em 440/490 and 540/580 nm)
  • Sample type- Adherent and suspension cells
  • Species reactivity- Mammalian
  • Applications- This assay provides a convenient and accurate procedure to measure de novo Protein synthesized in biological samples.

Detection Method

Flow Cytometry (Ex/Em 488/(530/590) nm) and Fluorescence Microscopy (Ex/Em 440/490 and 540/580 nm)

Species Reactivity

Eukaryotes

Applications

This assay provides a convenient and accurate procedure to measure de novo phopholipid synthesized in biological samples.

Features and Benefits

Simple, fast, does not require lengthy incubation times

Kit Components

  • Wash Buffer (10X)
  • Fixative Solution
  • Permeabilization Buffer (10X)
  • EZClickTM Phopholipid Label (100X)
  • Copper Reagent (100X)
  • Fluorescent Azide (100X)
  • Reducing Agent (20X)
  • Total DNA Stain (1000X)

Storage Conditions

-20°C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans

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