Collect, wash, and resuspend cells at ~1-2 x 10^6 cells/ml in RPMI 1640 media (no FBS). 2. Split samples, treat one with 100 ng/ml PTX, rotate at 37°C for 3-4 h (or 1 h without PTX). 3. Wash cells twice with assay buffer + 2.5 mM probenecid. 4. Resuspend cells in loading buffer (assay buffer + probenecid + 2.28 µg/ml Fluo-4 + 0.91 µg/ml Fura-red). 5. Protect tubes from light, rotate for 30 min at 37°C. If staining is incomplete, repeat the staining steps. 6. Wash cells, resuspend in assay buffer + probenecid at ~5 x 10^6 cells/ml. 7. For SDF-mediated calcium flux analysis, aliquot 450 µl cells into a FACS tube, record basal FITC/PerCP-Cy5.5 levels. 8. Stimulate cells with 50 µl 10 µg/ml SDF-1, record responses for 3 min, then stimulate with 50 µl 20 µM ionomycin. If responses are not clear, repeat the stimulation and recording steps. 9. Note: Replace SDF-1 with other chemokines as desired, determine optimal dose.