INDO-1 LOADING AND SAMPLE STAINING PROCEDURE FOR SIMULTANEOUS MEASUREMENT OF INTRACELLULAR CA2+ AND CELL SURFACE ANTIGEN EXPRESSION
MATERIALS:
1. 50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR)
2. DMSO (Sigma, St. Louis, MO)
3. RPMI 1640
4. Monoclonal antibodies (mAb), conjugated to suitable fluorochromes
5. Ionomycin (Calbiochem, San Diego, CA)
6. 37°C water bath, centrifuge, vortexer.
7. Agonists to test Ca2+ flux, e.g. anti-CD3, anti-IgG, ConA.
8. Serum (for RPMI with 2% serum, if cells require serum).
METHOD:
1. Incubate cells (£2x107/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.
2. 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 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC. Note: mAbs must be azide free. Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.
3. 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 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC.
4. Samples should be analyzed shortly after the cells were prepared.
5. Ionomycin (1-3mM final conc.) is used as a positive control for Indo-1 loading and maximum Ca2+ flux.
PREPARATION OF INDO-1:
1. Add 150 ml of DMSO to a 50 mg vial of Indo-1, cover with aluminum foil to protect from light.
2. Vortex well, then warm to 37°C for 5 min.
3. 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 –20oC for less than 6 months.
4. Cover the tube of 5 ml of 10 mM Indo-1 with foil.
5. 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.
6. 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:
1. Dissolve 1 mg of ionomycin in 1 ml DMSO.
2. Aliquot 13.5 ml of ionomycin solution into vials for later use and store at -20°C for less than one year.
3. Dilute one 13.5ml vial of ionomycin with RPMI to a volume of 3 mls (=6 mM).
4. Cover the 3 mls of 6 mM working stock with foil to protect from light.
5. 150 ml of working stock ionomycin is added to 300 ml of Indo-1 loaded cell suspension.
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.
Further Reading:
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.
1. 50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR)
2. DMSO (Sigma, St. Louis, MO)
3. RPMI 1640
4. Monoclonal antibodies (mAb), conjugated to suitable fluorochromes
5. Ionomycin (Calbiochem, San Diego, CA)
6. 37°C water bath, centrifuge, vortexer.
7. Agonists to test Ca2+ flux, e.g. anti-CD3, anti-IgG, ConA.
8. Serum (for RPMI with 2% serum, if cells require serum).
METHOD:
1. Incubate cells (£2x107/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.
2. 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 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC. Note: mAbs must be azide free. Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.
3. 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 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC.
4. Samples should be analyzed shortly after the cells were prepared.
5. Ionomycin (1-3mM final conc.) is used as a positive control for Indo-1 loading and maximum Ca2+ flux.
PREPARATION OF INDO-1:
1. Add 150 ml of DMSO to a 50 mg vial of Indo-1, cover with aluminum foil to protect from light.
2. Vortex well, then warm to 37°C for 5 min.
3. 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 –20oC for less than 6 months.
4. Cover the tube of 5 ml of 10 mM Indo-1 with foil.
5. 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.
6. 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:
1. Dissolve 1 mg of ionomycin in 1 ml DMSO.
2. Aliquot 13.5 ml of ionomycin solution into vials for later use and store at -20°C for less than one year.
3. Dilute one 13.5ml vial of ionomycin with RPMI to a volume of 3 mls (=6 mM).
4. Cover the 3 mls of 6 mM working stock with foil to protect from light.
5. 150 ml of working stock ionomycin is added to 300 ml of Indo-1 loaded cell suspension.
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.
Further Reading:
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.
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