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Cell Meter™ Fluorescence Gap Junction Tracing Kit

The GAP junctions were analyzed by flow cytometry. HeLa cells were stained with Calcein Ultragreen AM and DiD separately as per the protocol.  Cells were mixed well with 1:1 ratio and replated with cell culture medium. Response was measured using NovoCyte flow cytometer (ACEA Biosciences) with FITC and APC channel. As time progress, Q2-2 population (double positive population) increases.
The GAP junctions were analyzed by flow cytometry. HeLa cells were stained with Calcein Ultragreen AM and DiD separately as per the protocol.  Cells were mixed well with 1:1 ratio and replated with cell culture medium. Response was measured using NovoCyte flow cytometer (ACEA Biosciences) with FITC and APC channel. As time progress, Q2-2 population (double positive population) increases.
The GAP junctions were analyzed by fluorescence microscopy. HeLa cells were stained with Calcein Ultragreen AM and DiD separately as per the protocol.  Cells were mixed well with 1:1 ratio and replated with cell culture medium. Images were acquired using a fluorescence microscope with the FITC and Cy5 filter sets. As time progresses, double positive population of Calcein Ultragreen AM and DiD increases.
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Catalog Number23600
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Gap junctions are specialized intercellular membrane channels constituted with connexin that selectively facilitate the passage of small molecules of <1.5 kD across cells. They are tightly regulated by voltage, growth factors, cAMP, and retinoids, and they are modulated by phosphorylation. Cell Meter™ Fluorescence Gap Junction Tracing Kit provides a reliable and robust assay for the in vitro determination of gap junction function. The method is noninvasive. The cell population under study is divided such that one fraction is loaded with a lipophilic cell plasma membrane permeable dye, Calcein UltraGreen™ AM, that is hydrolyzed upon cellular uptake by cytoplasmic esterases to yield Calcein UltraGreen, a highly fluorescent and well-retained and membrane-impermeable molecule. The other fraction is loaded DiD, which is a lipophilic membrane dye that diffuses laterally to stain the entire cell membrane in deep red fluorescence upon incorporation into membranes. The two fractions are mixed and incubated under coculture conditions. Calcein UltraGreen is transferred to the DiD-stained cells through gap junctions. The assessment of this uptake can be monitored by fluorescence imaging or flow cytometry.

Platform


Flow cytometer

Excitation488 nm and 640 nm laser
Emission530/30 nm and 660/20 nm filters
Instrument specification(s)FITC and APC channel

Fluorescence microscope

ExcitationFITC and Cy5 filter sets
EmissionFITC and Cy5 filter sets
Recommended plateBlack wall/clear bottom

Components


Component A: Calcein UltraGreen™ AM2 vials
Component B: DiD1 vial
Component C: GAP Junction Assay Buffer1 bottle (100 mL)
Component D: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary
  1. Add Calcein Ultragreen AM working solution to the cells
  2. Add DiD working solution to the cells
  3. Incubate  at 37 °C for 10-30 minutes
  4. Wash cells with GAP Junction Assay Buffer
  5. Resuspend cells in cell culture medium and mix them with 1:1 ratio
  6. Measure the fluorescence signal at various times using  a flow cytometer with 530/30 nm and 660/20 nm emission filters or a fluorescence microscope with FITC and Cy5 filter sets 

Important
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Calcein Ultragreen AM stock solution
Add 50 µL of DMSO (Component D) into one Calcein Ultragreen AM vial (Component A) and mix well.
Note     Store the unused Calcein Ultragreen AM stock solution at -20 °C in single use aliquots to avoid freeze thaw cycles.
Note     One vial is enough for 50 tests.


2. DiD stock solution
Add 100 µL of DMSO (Component D) into DiD (Component B) and mix well.
Note     Store the unused DiD stock solution at -20 °C in single use aliquots to avoid freeze thaw cycles.

PREPARATION OF WORKING SOLUTION

1. Calcein Ultragreen AM working solution
Add 1 µL of Calcein Ultragreen AM stock solution to 1 mL of GAP Junction Assay Buffer and mix well.
Note     Calcein Ultragreen AM working solution should not be stored and should be used promptly.
Note     1 mL Calcein Ultragreen AM working solution is enough for two tests.


2. DiD working solution
Add 1 µL of DiD stock solution into 1 mL of GAP Junction Assay Buffer and mix well.
Note     DiD working solution should not be stored and should be used promptly.
Note     1 mL DiD working solution is enough for two tests.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol can be used as a guideline and should be optimized according to the needs.

Cell staining protocol for Calcein Ultragreen AM
  1. Grow cells in cell culture medium in 6-well cell culture plates.
  2. Remove the cell culture medium and add 0.5 mL of Calcein Ultragreen AM working solution.
  3. Incubate cells at 37 °C for 10-20 minutes.
    Note     Incubation time should be optimized for each cell line.
  4. Remove the dye working solution and wash cells with GAP Junction Assay buffer.
    Note     For the adherent cells, detach cells from the plate using rubber policeman.
  5. Resuspend cells in cell culture medium. 

Cell labelling protocol for DiD
  1. Grow cells in cell culture medium in 6-well cell culture plates.
  2. Remove the cell culture medium and add 0.5 mL of DiD working solution.
  3. Incubate cells at 37 °C for 10-20 minutes.
    Note     Incubation time should be optimized for each cell line.
  4. Remove the dye working solution and wash cells with GAP Junction Assay Buffer.
    Note     For the adherent cells, detach cells from the plate using rubber policeman.
  5. Resuspend cells in cell culture medium. 

GAP junction assay
  1. Mix Calcein stained cells and DiD labelled cells with 1:1 ratio and plate them in wells.
    Note     For the fluorescence microscopy, add 50 µL of each into the well of a 96-well plate and mix well.
    Note     For the flow cytometer, add 500 µL of each into the well of a 6-well plate and mix well.
  2. Incubate cells at 37 °C for 2-3 hours.
  3. Monitor the cells with a fluorescence microscope using the FITC and Cy5 filter sets.
  4. For flow cytometric analysis: For adherent cells, detach cells using rubber policeman. Once cells are in suspension or for cells in suspension, wash cells twice with DPBS or buffer of your choice.  Resuspend cells in HHBS (cat# 20011), or DPBS or buffer of your choice and measure response with 530/30 nm filter (FITC channel) and 660/20 nm filter (APC channel). 

References


View all 50 references: Citation Explorer
Regulation of connexin 43 by interleukin 1β in adult rat cardiac fibroblasts and effects in an adult rat cardiac myocyte: fibroblast co-culture model.
Authors: McArthur, Lisa and Riddell, Alexandra and Chilton, Lisa and Smith, Godfrey L and Nicklin, Stuart A
Journal: Heliyon (2020): e03031
Cilostamide and forskolin maintain gap junction function of incubated dog follicles.
Authors: Thongkittidilok, Chommanart and Doriguzzi, Nicole and Nagashima, Jennifer and Brown, Megan and Chansaenroj, Ajjima and Songsasen, Nucharin
Journal: Theriogenology (2020): 222-228
Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.
Authors: Rouhollahi Varnosfaderani, Shiva and Hajian, Mehdi and Jafarpour, Farnoosh and Ghazvini Zadegan, Faezeh and Nasr-Esfahani, Mohammad Hossein
Journal: PloS one (2020): e0229043
G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17β-estradiol.
Authors: Zhang, Hui and Wei, Qiang and Gao, Zhen and Ma, Chiyuan and Yang, Zhenshan and Zhao, Hui and Liu, Chen and Liu, Jie and Zhao, Xiaoe and Ma, Baohua
Journal: The Journal of steroid biochemistry and molecular biology (2019): 58-67
An Assay to Assess Gap Junction Communication in Cell Lines.
Authors: Warawdekar, Ujjwala M
Journal: Journal of biomolecular techniques : JBT (2019): 1-6
Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.
Authors: Hofmann, Francisca and Navarrete, Mariela and Álvarez, Javiera and Guerrero, Israel and Gleisner, María Alejandra and Tittarelli, Andrés and Salazar-Onfray, Flavio
Journal: International journal of molecular sciences (2019)
Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.
Authors: Schultz, Francisca and Swiatlowska, Pamela and Alvarez-Laviada, Anita and Sanchez-Alonso, Jose L and Song, Qianqian and de Vries, Antoine A F and Pijnappels, Daniël A and Ongstad, Emily and Braga, Vania M M and Entcheva, Emilia and Gourdie, Robert G and Miragoli, Michele and Gorelik, Julia
Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2019): 10453-10468
Perfluorooctanoic acid disrupts gap junction intercellular communication and induces reactive oxygen species formation and apoptosis in mouse ovaries.
Authors: López-Arellano, Patricia and López-Arellano, Keila and Luna, Jaquelinne and Flores, Diana and Jiménez-Salazar, Javier and Gavia, Graciela and Teteltitla, Mario and Rodríguez, Juan José and Domínguez, Alejandro and Casas, Eduardo and Bahena, Ivan and Betancourt, Miguel and González, Cristina and Ducolomb, Yvonne and Bonilla, Edmundo
Journal: Environmental toxicology (2019): 92-98
Response of Fibroblasts MRC-5 to Flufenamic Acid-Grafted MCM-41 Nanoparticles.
Authors: Lara, Giovanna Gomes and Cipreste, Marcelo Fernandes and Andrade, Gracielle Ferreira and Silva, Wellington Marcos da and Sousa, Edésia Martins Barros de
Journal: Bioengineering (Basel, Switzerland) (2018)
Heat stress impairs gap junction communication and cumulus function of bovine oocytes.
Authors: Campen, Kelly A and Abbott, Chelsea R and Rispoli, Louisa A and Payton, Rebecca R and Saxton, Arnold M and Edwards, J Lannett
Journal: The Journal of reproduction and development (2018): 385-392