Hi Sebastien,
Looking again at your initial flow plots slide, I want to confirm a couple more things.
1. You took whole blood, did ACK lysis.
2. Since you used the Fluidigm barcoding kit, the cells were then Fixed and Perm'd as on page 7 of the User Guide?
http://www.dvssciences.com/products/pdf/UG_201060.pdf3. You then did a single antibody stain separately on each sample. Your samples were:
Sample 5_P1.FCS (top of slide): Barcoded with 102/104/105. Antibody stained with *only* 89Y-CD45 (not CD3-170Er or CD45-141Pr). Stained with Ir. No live-dead (cisplatin, etc).
Sample 7_P3_01.fcs (middle of slide): Barcoded with 102/104/108. Antibody stained with *only* CD3-170Er (not 89Y-CD45 or CD45-141Pr). Stained with Ir. No live-dead (cisplatin, etc).
Sample 6_P2_01.fcs (bottom of slide): Barcoded with 102/104/106. Antibody stained with *only* CD45-141Pr (not 89Y-CD45 or CD3-170Er). Stained with Ir. No live-dead (cisplatin, etc).
-By the way: I assume it was CD45-Pr141 as you wrote in your original post. Your flow plots say CD196-141Pr
4. Then Ir staining, and running on the machine.
In other words, each samples was fixed, then perm'd, stained with a Barcode, stained with *one* antibody each (not a cocktail of 3 antibodies as I wrote in my initial reply yesterday), and then stained with Ir. Is this correct?
Mike