Materials and Reagents

Cells of choice
RPMI 1640 media (Life Technologies, Invitrogen™, catalog number: 11875-119 )
Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 10437-028 )
PTX (List Biological Labs, catalog number: 183 )
HBSS (Life Technologies, Invitrogen™, catalog number: 14185-052 )
BSA (Sigma Aldrich, catalog number: A7030 )
HEPES (Life Technologies, Invitrogen™, catalog number: 15630-080 )
Probenecid (Sigma Aldrich, catalog number: P8761 )
Fluo-4 (Life Technologies, Invitrogen™, catalog number: F14201 )
Fura-red (Life Technologies, Invitrogen™, catalog number: F3021 )
Pluronic F-127 (Life Technologies, Invitrogen™, catalog number: P-3000MP )
DMSO (Sigma Aldrich, catalog number: D2650 )
Ionomycin (Sigma Aldrich, catalog number: I0634 )
NaOH (Thermo Fisher Scientific, catalog number: BP359-212 )
Assay buffer (see Recipes)
Loading buffer (see Recipes)

Equipment

Procedure

Cells are collected, washed and resuspended at ~1-2 x 10⁶ cells/ml in RPMI 1640 media (no FBS).
If treated with PTX, then samples are split in 2 with one receiving 100 ng/ml PTX and the other PBS. Tubes are rotated at 37 °C for 3-4 h.
If cells are not pretreated with PTX, then cells are rotated for 1 h at 37 °C.
Cells are then washed twice with assay buffer+ 2.5 mM probenecid.
Resuspend cells in loading buffer (assay buffer + probenecid + 2.28 µg/ml Fluo-4 + 0.91 µg/ml Fura-red) at ~4 x 10⁶ cells/ml. N.B. In both cases, 50 µg of Fluo-4 and Fura-red are dissolved in 22 µl pluronic F-127 (20% solution in DMSO) and 22 µl DMSO.
Light protected tubes are rotated for 30 min at 37 °C.
Cells are then washed once and resuspended with assay buffer + probenecid at ~ 5 x 10⁶ cells/ml.
For SDF-mediated calcium flux analysis, 450 µl of cells are aliquotted into a FACS tube and basal ratio of FITC/PerCP-Cy5.5 levels are recorded. At 1 min, cells are stimulated with 50 µl 10 µg/ml (~125 nM final) SDF-1 and responses recorded for a further 3 min. Finally, cells are stimulated with 50 µl 20 µM ionomycin.
Note: SDF-1 can be replaced by other chemokines as you desire. However, optimal dose needs to be determined.

Notes

Assay buffer can be stored at 4 °C. Allow to reach room temperature before use.
Probenecid is prepared fresh each time in 1 N NaOH (stock = 500 mM).
Unused Fluo-4 and Fura-Red, once reconstituted in DMSO/Pluronic acid, can be refrozen once for use on a future date.

Recipes

Acknowledgments

This protocol was adapted from previously published reports (Cronshaw et al., 2006 and Cronshaw et al., 2010).

References