Calcium flux:  Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+

doi:10.1038/nprot.2007.66

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biochemistry

Computational biology and bioinformatics

Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca²⁺

June Round

UCLA

DOI:

10.1038/nprot.2007.66

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

This protocol was used in the above Nature Immunology paper.

50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR)
DMSO (Sigma, St. Louis, MO)
RPMI 1640
Monoclonal antibodies (mAb), conjugated to suitable fluorochromes
Ionomycin (Calbiochem, San Diego, CA)
37°C water bath, centrifuge, vortexer.
Agonists to test Ca²⁺ flux, e.g. anti-CD3, anti-IgG, ConA.
Serum (for RPMI with 2% serum, if cells require serum).

PREPARATION OF INDO-1:

Add 150 ml of DMSO to a 50 mg vial of Indo-1, cover with aluminum foil to protect from light.
Vortex well, then warm to 37°C for 5 min.
Transfer 150 ml of Indo-1 from vial to 4.85 mls of RPMI (=10mM). Wash out vial very well. If not the entire amount of Indo-1 dissolved in DMSO is used, the remainder can be stored dessicated at –20°C for less than 6 months.
Cover the tube of 5 ml of 10 mM Indo-1 with foil.
Aliquot the appropriate amount of 10mM Indo-1 to the cell suspension (final conc.=1-5 mM). The optimal concentration is dependent on the cell type.
Store excess RPMI-diluted 10 mM Indo-1 at 4°C. In our laboratory, the 10 mM Indo-1 solution has been tested for stability up to 24 hrs.

PREPARATION OF IONOMYCIN:

Dissolve 1 mg of ionomycin in 1 ml DMSO.
Aliquot 13.5 ml of ionomycin solution into vials for later use and store at -20°C for less than one year.
Dilute one 13.5ml vial of ionomycin with RPMI to a volume of 3 mls (=6 mM).
Cover the 3 mls of 6 mM working stock with foil to protect from light.
150 ml of working stock ionomycin is added to 300 ml of Indo-1 loaded cell suspension.

Incubate cells (2x10⁷/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.

Incubate aliquots of Indo-1 loaded cells with saturating concentrations of e.g., FITC, PE, PerCP, or Tricolor-conjugated antibodies for 20 min. Incubate at 20° to 25 C unless the antigen is subject to capping, otherwise use 4° to 8 C. Note: mAbs must be azide free.

Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.

Wash cells twice in RPMI and suspend them at the desired concentration (usually 2x10⁶/ml). Higher cell concentrations (4x10⁶/ml) are required when the cells of interest represent less than 10% of the total population. Cells can be kept at 20° to 25 C unless the antigen is subject to capping, otherwise use 4° to 8 C.

Samples should be analyzed shortly after the cells were prepared.

Ionomycin (1-3mM final conc.) is used as a positive control for Indo-1 loading and maximum Ca²⁺ flux.

Special Note:

Indo-1 requires UV excitation. Make sure that you have an instrument with either an argon laser tuned to UV or a helium-cadmium laser available. Because experiments involving the measurement of calcium flux are performed directly on the flow cytometer, pre experiment consultation is strongly recommended.

June CH, Rabinovitch PS. Intracellular ionized calcium. Methods Cell Biol. 1994; 41 :149-74.

June CH, Abe R, Rabinovitch PS. Measurement of intracellular calcium ions by flow cytometry. In: Current Protocols in Cytometry, Vol 2, Robinson JP, Darzynkiewicz Z, Hyun W, Orfao A, Rabinovitch P, eds., John Wiley & Sons, 1997, pp. 9.8.1 – 9.8.19.

Gayatri Venkiteswaran

commented on 29 February, 2008

I have tried to do indo-1 imaging on some neuronal cultures.the concentration of the dye that i had used for loading was 5uM. when i add ionomycin to the cells, i not only see an increase in fluorescence signal at 405 nm but also a small increase at 485 nm.I don`t know if this is due to excess dye in the cell. Can i have u`r suggestions on this ? Thanks. Gayatri. (India)

commented on 25 January, 2011

1) The DMSO volume should be 150 micor liter, not ml. 2) I usually 5 uM Indo-1 in PBMC and I could get fairly good signals with flow cytometry. 3) Invitrogen does not have 50 mg Indo-1 or it has been discontinued, and I doubt it ever had. The cat# is corresponding to 1mg vial. Thanks, Zhifei

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Version History

CURRENT STATUS: Posted

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Subject Areas

Biochemistry

Computational biology and bioinformatics

Associated Publications

Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NFkappa-B transcription factors
by Round, J.L. et al.
Nature Immunology (25 January, 2007)

Method Article

Biochemistry

Computational biology and bioinformatics

Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca²⁺

June Round

UCLA

Version History

CURRENT STATUS: Posted

Version 1

Posted 29 Jan, 2007

MetricsComments: 4HTML Views: ...

Subject Areas

Biochemistry

Computational biology and bioinformatics

DOI:

10.1038/nprot.2007.66

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

This protocol was used in the above Nature Immunology paper.

50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR)
DMSO (Sigma, St. Louis, MO)
RPMI 1640
Monoclonal antibodies (mAb), conjugated to suitable fluorochromes
Ionomycin (Calbiochem, San Diego, CA)
37°C water bath, centrifuge, vortexer.
Agonists to test Ca²⁺ flux, e.g. anti-CD3, anti-IgG, ConA.
Serum (for RPMI with 2% serum, if cells require serum).

PREPARATION OF INDO-1:

Add 150 ml of DMSO to a 50 mg vial of Indo-1, cover with aluminum foil to protect from light.
Vortex well, then warm to 37°C for 5 min.
Transfer 150 ml of Indo-1 from vial to 4.85 mls of RPMI (=10mM). Wash out vial very well. If not the entire amount of Indo-1 dissolved in DMSO is used, the remainder can be stored dessicated at –20°C for less than 6 months.
Cover the tube of 5 ml of 10 mM Indo-1 with foil.
Aliquot the appropriate amount of 10mM Indo-1 to the cell suspension (final conc.=1-5 mM). The optimal concentration is dependent on the cell type.
Store excess RPMI-diluted 10 mM Indo-1 at 4°C. In our laboratory, the 10 mM Indo-1 solution has been tested for stability up to 24 hrs.

PREPARATION OF IONOMYCIN:

Dissolve 1 mg of ionomycin in 1 ml DMSO.
Aliquot 13.5 ml of ionomycin solution into vials for later use and store at -20°C for less than one year.
Dilute one 13.5ml vial of ionomycin with RPMI to a volume of 3 mls (=6 mM).
Cover the 3 mls of 6 mM working stock with foil to protect from light.
150 ml of working stock ionomycin is added to 300 ml of Indo-1 loaded cell suspension.

Incubate cells (2x10⁷/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.

Incubate aliquots of Indo-1 loaded cells with saturating concentrations of e.g., FITC, PE, PerCP, or Tricolor-conjugated antibodies for 20 min. Incubate at 20° to 25 C unless the antigen is subject to capping, otherwise use 4° to 8 C. Note: mAbs must be azide free.

Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.

Wash cells twice in RPMI and suspend them at the desired concentration (usually 2x10⁶/ml). Higher cell concentrations (4x10⁶/ml) are required when the cells of interest represent less than 10% of the total population. Cells can be kept at 20° to 25 C unless the antigen is subject to capping, otherwise use 4° to 8 C.

Samples should be analyzed shortly after the cells were prepared.

Ionomycin (1-3mM final conc.) is used as a positive control for Indo-1 loading and maximum Ca²⁺ flux.

Special Note:

June CH, Rabinovitch PS. Intracellular ionized calcium. Methods Cell Biol. 1994; 41 :149-74.

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Associated Publications

Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NFkappa-B transcription factors
by Round, J.L. et al.
Nature Immunology (25 January, 2007)

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biochemistry

Computational biology and bioinformatics

Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+

June Round

June Round

UCLA

DOI:

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Introduction

Reagents

Procedure

Critical Steps

References

Comments (4)

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Version History

CURRENT STATUS: Posted

Version 1

Metrics

Comments: 4

HTML Views: ...

Subject Areas

Biochemistry

Computational biology and bioinformatics

Associated Publications

Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NFkappa-B transcription factors

by Round, J.L. et al.

Nature Immunology (25 January, 2007)

Method Article

Biochemistry

Computational biology and bioinformatics

Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+

June Round

June Round

UCLA

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License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Declarations

Introduction

Reagents

Procedure

Critical Steps

References

Comments (4)

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Gayatri Venkiteswaran

commented on 29 February, 2008

commented on 25 January, 2011

Related Articles

Associated Publications

Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NFkappa-B transcription factors

by Round, J.L. et al.

Nature Immunology (25 January, 2007)

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Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca²⁺

Calcium flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca²⁺