I'm trying to transfect primary PBMCs with siRNA in a Lonza 4-D Nucleofector, but I've had no luck. So far, I've been using Lonza's Amaxa unstimulated T-cell protocols, in the 20 uL/well 2x8-well strip.

• RPMI-1640 used for culture, 200k-250k cells per well in a 96 well plate (density: 1 M cells/mL). Perform nucleofection 24 hrs post-thaw.
• Collect cells into 50 mL tube. Count via hemocytometer. Distribute 5-10 M cells worth to 15 mL tubes. Centrifuge at 200 x g for 10 minutes to collect cells, carefully vacuum off supernatant.
• Resuspend in at least 25 uL P3, layer over 1 uL siRNA /(2 ug pmaxGFP), gently mix with P200 (no bubbles!).
• Transfer to marked locations on the 2x8 strip with p10, two 10uL volumes. Hold at angle when dispensing and tap bottom to prevent air bubbles. Put in nucleofector in the correct orientation.
• Mark wells and use code: EO-115 for high efficiency.
• Let sit for 10 minutes at room temperature. Transfer contents to respective microcentrifuge tubes. Distribute to new pre-warmed culture plate, 500k cells per well ( 2 M cells/mL, assuming 100% viability post-nucleofection).
• Wait 24 hours, check GFP under fluorescent scope (up to 40x).

All my reagents are new and unexpired, but I don't get the type of confirmatory fluorescence from my PBMCs like our lab does with keratinocytes or MSCs. Does anyone have any experience with this? Am I doing something wrong, or is it because PBMCs are too small to see a difference in GFP signal?