BD Pharmingen™ Calcein AM

Preparation

Bring BD Pharmingen™ Calcein AM powder and fresh cell culture-grade Dimethyl Sulfoxide (DMSO; e.g., Sigma, Cat. No. D2650) to room temperature. Add 201 μl of DMSO to make a 5 mM stock solution (Molar Mass = 994.86 g/mol) and vortex solution well. Inspect the solution and repeat vortex until the stock dye has fully dissolved.

Storage

Store BD Pharmingen™ Calcein AM desiccated at -80°C until use. After reconstitution with DMSO, store the solution at -20°C in small aliquots. Avoid freeze-thaw cycles. Please discard the dye aliquots after 6 months post reconstitution with DMSO.

Notes

•        When diluted with an aqueous solution, BD Pharmingen™ Calcein AM should be used immediately after dilution, as the esterase-cleavable dye may spontaneously hydrolyze with prolonged aqueous exposure.

•        Please note that these dyes are esterase cleavable. Serum may have esterase activity, and serum-containing buffers may decrease the resolution of viable and non-viable cells if used during staining. Serum-containing buffers may be used for wash steps after staining is complete.

•        The spillover properties of this dye are similar to FITC. As such, we recommend appropriate compensation for other colors excited by the blue laser, such as PE or PerCP-Cy™5.5. Additionally, we recommend using cells stained with BD Pharmingen™ Calcein AM for compensation.

•        This dye is not fixable.

General Procedure

1.        Prepare cells for flow cytometry staining using serum-free buffer (e.g., 1X Dulbecco's Phosphate Buffered Saline).

2.        Wash cells one time in serum-free buffer.

3.        Resuspend cells at ~1-10 x 10^6 cells/ml in dye diluted in serum-free buffer.

a.     We recommend staining at a 1-10 μM final concentration of BD Pharmingen™ Calcein AM dye for live/dead cell discrimination, or at 0.1-1 μM for multicolor applications as a starting point. Since fluorescence intensity may vary based on cell type and experimental conditions, a titration is recommended to determine the optimal dye concentration for each experimental protocol.

4.        Incubate the mixture for 30 minutes at room temperature protected from light.

5.        Wash cells twice with 2 ml of BD Pharmingen™ Stain Buffer (FBS) or the equivalent.

6.        Aspirate the supernatant and gently mix to disrupt the cell pellet.

7.        Resuspend the cells in Stain Buffer (FBS) or equivalent.

8.        Analyze by flow cytometry or stain cells as desired for downstream applications.

Notes on Adherent Cells

Adherent cells may be stained in suspension or in situ.

To stain adherent cells in suspension, follow the "General Procedure" as outlined above after cell dissociation into a single cell suspension. Note that for some cells, enzymatic removal from the growth substrate may impact cell membrane integrity, which may affect staining with BD Pharmingen™ Calcein AM. Allowing the cells to recover from the enzymatic dissociation in growth media is recommended. The time of recovery may vary by cell line, so it may be useful to test multiple recovery periods in initial experiments.

To stain in situ, we recommend the following protocol:

1.        Remove media and wash the cells once with serum-free buffer to remove any remaining media.

2.        Add dye diluted in serum-free buffer to the culture.

a.        We recommend staining at a 1-10 μM concentration of BD Pharmingen™ Calcein AM dye for live/dead cell discrimination, or at 0.1-1 μM for multicolor applications. Since brightness may vary based on cell type and experimental conditions, a titration is recommended to determine the optimal dye concentration for each experimental protocol.

3.        Incubate the mixture for 30-60 minutes at 37°C protected from light.

4.        Wash cultures twice with BD Pharmingen™ Stain Buffer (FBS) or the equivalent.

5.        Remove the cells from the growth substrate. We recommend using BD™ Accutase™ Cell Detachment Solution.

a.        Wash cells twice with BD Pharmingen™ Stain Buffer (FBS) or the equivalent.

b.        Resuspend the cells in Stain Buffer (FBS) or equivalent.

c.        Analyze by flow cytometry or stain cells as desired for downstream applications.

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
3. Accutase is a registered trademark of Innovative Cell Technologies, Inc.
4. All other brands are trademarks of their respective owners.
5. Cy is a trademark of GE Healthcare.
6. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.