Guava 6ht flow cytometer

Multiplex miRNAs were measured using a Firefly miRNA particle assay system coupled with a portable flow cytometer/reader (Guava^® easyCyte™ 6HT, Millipore, Burlington, MA). Sixty-eight neurological miRNA targets were screened using FirePlex™ miRNA neurology panel V2 (cat# ab218371, Abcam, Waltham, MA). Briefly, according to the protocol from the manufacturer, RNA samples were extracted by incubating 20 ul serum, 20 ul RNase-free water, and 40 ul Lysis Mix for 45 min at 60°C while shaking. Then, in a 96-well filter plate, the Firefly miRNA kit was incubated with 25 ul Hybridization Buffer and 25 ul extracted RNA at 37°C for 60 min. After rinsing to remove unbound RNA, 75 ul of Labeling Buffer was added to each well, and the plate was incubated for 60 min at room temperature. Adapted-modified miRNAs were released from the particles by incubating with Rnase-free water for 30 min at 62.5°C, and PCR amplified using a fluorescently-labeled primer set. The PCR product was hybridized to fresh Firefly particles at 37°C for 30 min and re-suspended in Run Buffer for readout. Particles were scanned on an EMD Millipore Guava 6HT flow cytometer. The raw output was background subtracted, normalized using the geometric mean of the normalizer miRNAs, and log transformed.

After infection, the well contents were collected and centrifuged at 1,000 × g for 2 min. The cell pellet was washed twice with flow buffer (2% FBS in PBS) and resuspended in 50 μl of flow buffer for staining. Neutrophils were stained for 25 min with CD63-PE-Cy7 (Invitrogen) on ice. Neutrophils were washed once in flow buffer and resuspended in flow buffer containing propidium iodide (Invitrogen) to exclude dead cells. Neutrophils were acquired using a Millipore Guava 6HT flow cytometer and analyzed with the InCyte EasyCyte v3.1 software.