Ambion single cell to ct qrt pcr kit

Single cells were captured and the loci of interest preamplified using the C1 system (Fluidigm, software version 2.2.1) following the manufacturer's protocol. Cells were partitioned using a 10–17 μm C1 Single-Cell Preamp integrated fluidic circuit (IFC) (PN 100-5479; Fluidigm). IFC priming, cell loading and lysis, reverse transcription, and preamplification were then carried out using reagents from the following kits: C1 Single-Cell Auto Prep Reagent Kit (PN 100-5139; Fluidigm), Ambion Single Cell-to-CT qRT-PCR Kit (4458237; Thermo Fisher Scientific), and 20X TaqMan Gene Expression primers (Thermo Fisher Scientific). Amplicons were transferred from the IFC to a 96-well plate and stored at −20°C.

Cells were counted using an automated counter (TC-20, Bio-Rad, Hercules, CA) and manual counting using a hemocytometer. Cells were washed twice in PBS and resuspended at a concentration of 1200 cells/µL. Cells (6 µL) and reagents (Ambion Single cell-to-CT qRT-PCR kit, Thermo Fisher, Waltham CA) were loaded into the C1 integrated fluidic circuits (IFCs) as per manufacturer’s recommendations.
Validations were performed to determine optimal cell concentration, cell buoyancy, and input for highest single-cell capture rate. Cell capture was evaluated by microscopy directly following ‘Cell Load’ script. Single, healthy-appearing cells were selected for downstream analysis post-C1 protocol. Cell lysis, reverse transcription and cDNA pre-amplification were carried out using the C1 instrument (Fluidigm Corp., San Francisco, CA).
The IFC size selected was based on expected cell size: 5-10 µm chips were used for unstimulated J-Lat (clone 9.2), 8E5, U1 and ACH-2, whereas 10-17 µm IFC were selected for stimulated J-Lat cells. Following C1 assay completion, cDNA was harvested and diluted 1 in 8 in C1 DNA Dilution buffer (Fluidigm Corp.) and stored at − 20 °C prior to use.