High-throughput microwell high-dimensional fluorescence activated Single cell sorting (HT-μW-HiD-FASCS)

Dr. Kartoosh Heydari

doi:/

Volume : 1 | Issue : 1

Technical Note

High-throughput microwell high-dimensional fluorescence activated Single cell sorting (HT-μW-HiD-FASCS)

Kartoosh Heydari

Director of Li Ka Shing Center FACS Core Facility, Department of Molecular Cell Biology / Cancer Research Laboratory, UC Berkeley, CA

Received: February 02, 2018 | Published:February 15, 2018

Correspondence:Dr. Kartoosh Heydari, Director of Li Ka Shing Center FACS Core Facility, Department of Molecular Cell Biology / Cancer Research Laboratory, UC Berkeley, CA, Tel 6503877646, Email kartooshheydari@berkeley.edu

Copyright© 2018 Heydari. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

High-throughput screening (HTS) and isolation of single cell, in clonal level, for biochemical, genomic, epigenomic, transcriptomic, proteomic, or metabolomic studies has beenrecently addressed by many scientific communities in field of Cancer Immuno-biology/-therapy and Regenerative Medicine.Fluorescence Activated Cell Sorting (FACS) has been the most innovative technology in life sciences and cell therapy since its invention in 1969 by my former postdoctoral advisers at Stanford University, ProfessorL. A. Herzenbergs, who received the Kyoto prize (Japanese equivalent to Nobel Prize) in Advanced Technology in 2006. Using theinnovative capacity of High-DimensionalFACS (HiD-FACS) and HiD-Fluorescence Activated Single Cell Sorting (HiD-FASCS), we have added a novel concept in cell biology and regenerative medicine fields by describing a novel cell subset with unique function we named “Privileged Cells”¹. We have now extended this innovative capacity of FASCS by {A} high-throughput(HT) single cell sorting (2000 wells/slid) in microWell (μW)(100 μm)(Figures 1–5 and Table 1), and {B} using high-resolution optical lens to improve the precision and the sensitivity of μW sort (Figure 4). HT-μW-HiD-FASCS is the most innovative technology for clonal level studies of the fine subsets of cells.

<strong>Figure 1:</strong>From High Throughput (96 well and Terasaki, 72 well) plate to HT (2000 micro-well) slide.

Figure 1:From High Throughput (96 well and Terasaki, 72 well) plate to HT (2000 micro-well) slide.

<strong>Figure 2: </strong>Development of High-Throughput (HT) microWell (μW) Fluorescence Activated Single Cell Sorting (FASCS)<strong>.</strong>

Figure 2: Development of High-Throughput (HT) microWell (μW) Fluorescence Activated Single Cell Sorting (FASCS).

<strong>Figure 3: </strong>Increase accuracy of single cells deposition on slide by avoiding air currents in the flow chamber, creating a vertical trajectory, re-positioning the waste stream catcher off center, and positioning the slide closer to the sorter nozzle.

Figure 3: Increase accuracy of single cells deposition on slide by avoiding air currents in the flow chamber, creating a vertical trajectory, re-positioning the waste stream catcher off center, and positioning the slide closer to the sorter nozzle.

<strong>Figure 4:</strong> Direct verification of successful drop deposition by inverted microscope with high-resolution optical lens: Andonstar Digital Microscope Video. Magnification: +100X, DSP: High Power Digital Image Monarch Processor; Sensor: high-quality CMOS sensor, <strong>2M pixel</strong>; Resolution: 640x480,1600x1200; Frame rate: 640x480 resolution=30 frames/sec,1600x1200=5frames/sec; Focal distance: 5mm–30mm.

Figure 4: Direct verification of successful drop deposition by inverted microscope with high-resolution optical lens: Andonstar Digital Microscope Video. Magnification: +100X, DSP: High Power Digital Image Monarch Processor; Sensor: high-quality CMOS sensor, 2M pixel; Resolution: 640x480,1600x1200; Frame rate: 640x480 resolution=30 frames/sec,1600x1200=5frames/sec; Focal distance: 5mm–30mm.

<strong>Figure 5:</strong> Single bacterial sorted on agar plate to verify the feasibility of HT-μW-HiD-FASCS.

Figure 5: Single bacterial sorted on agar plate to verify the feasibility of HT-μW-HiD-FASCS.

Instrument setup	Sample Preparation
Nozzle choice / sheath pressure Laser alignment and delay (Calibration beads) Drop delay adjustment (Accudrop beads) Gating strategy to gate out noise, debris and doublets Fsc / Ssc in log (cells & organelles < 1 um) Sort precision (single cell) Flow rate (for single cell <100 cell/s) Collection vessel targeting Sort Process	Single cell suspension (2-5% protein, EDTA 2-5mM) Samples with lots of dead cells (use DNAse, no EDTA) Match the cell concentration to the instrument setup and cell type Stain cocktail should be fully worked up prior to sorting Proper single color control (beads better than cells)

Table 1: Key Criteria for a successful optimization of sort setting for HT-μW-HiD-FASCS.

Vaccines and Immunology Open Access Journal (VIOAJ)

Open Access Journal

Frequency: Tri-Monthly

ISSN 2630-9270

High-throughput microwell high-dimensional fluorescence activated Single cell sorting (HT-μW-HiD-FASCS)

References