We are performing a one-way "MLR" using CSFE-labeled huPBMCs stimulated with CD3e and CD28 Abs to determine immunosuppression on huBM-MSCs. We are observing what appears to be non-specific suppression of activation and are concerned that the effect may be due to depletion of essential nutrients by MSCs.

The assay set up is:

• 96 well plate containing 150 uL of media/well
• MSCs (20 or 40 x 104) are plated in complete MSC medium
• after culturing MSCs overnight, the medium is replaced with RPMI + 10%FBS
• 2x105 PBMCs are added and stimulated with CD3e and CD28 Abs (each T concentrations optimized for activation)
• plates are incubated for 4 days without media change before flow analysis to detect activated T cell progeny

Supplementing with GlutaMax and glucose does not alleviate suppression.

Partially replacing the spent medium with complete medium containing Abs causes a loss of suppression. This effect is observed with as little as 20 uL medium changes daily.

There are many publications describing optimization of T cell suppression assays but none that we have found address this potential artefact.

We are looking for guidance on how to address this potential issue.