Instrumentation

Automated Microscopes

Yokogawa CV8000 (“Apollo”)

Apollo is one of two HiTIF’s high-throughput confocal spinning disk microscopes, which accepts any SBS-compatible imaging plate designed for high-content imaging (Mostly in 96- or 384-well format). It is equipped with 4 excitation laser lines (405, 488, 561, and 640 nm) and 2 sCMOS cameras (QE 82%, 2048 × 2048 pixels). Apollo has 6 objectives, including a 40X water objective with high numerical aperture (NA 1.15), and a 60X water objective (NA 1.2). In addition, Apollo can run automated long term live cell experiments for up to 72 hrs. Apollo is not currently integrated with a robotic arm.

Nikon Ti2 automated wide-field microscope

The Nikon Ti2 is a fully automated wide-field microscope equipped with 7 LED illumination lines, custom interchangeable excitation/emission filter sets, and a Digital Micromirror Device (DMD) unit for fully automated, spatially resolved (< 5 microns) photoactivation. Its wider excitation/emission range supports in situ sequencing (ISS) and spatially resolved photoactivation for pooled HCI assay development and screens. Use of the Ti2 is currently limited to HiTIF personnel for assay-development purposes and to a limited number of collaborators trained in pooled HCI.

Automated Liquid Handling

Using a combination of the liquid handlers below, the HiTIF can process up to 15 384-well plates per day for semi-automated PFA fixation, immunofluorescence (IF), and RNA/DNA FISH protocols. All liquid handlers, with the exception of the Bravo, are covered by service contracts.

Agilent Bravo Automated Liquid Handling Platform

The Agilent Bravo was inherited from the CCR Genomics Core. It is used to resuspend and reformat lyophilized RNA oligo libraries in 96- or 384-well storage plates, supporting downstream library management and assay-plate preparation.

Beckman ECHO525 Acoustic Liquid Handler

The ECHO525 uses focused sound waves to transfer liquids from any well of a source plate to any well of a destination plate in 25 nl increments. Source plates must be ECHO compatible (6-well reservoir, 384-well polypropylene, 384-well low dead volume). Destination plates can be in any format, provided they respect SBS standards and are not taller than 16 mm. The ECHO525 is used in library reformatting/management, siRNA or sgRNA oligos transfection optimization, and automated reverse transfection screening. In addition, the ECHO525 can be used for setup of qRT-PCR reactions, or synthetic biology assembly reactions in 384-well plates.

Biotek EL406 Plate washer/Dispenser

The Biotek EL406 Plate washer can be programmed for automated and semi-automated IF and DNA FISH protocols. It is equipped for with a washer module, a peristaltic pump based cassette dispenser and with two syringe pumps dispensers. If desired, plates can be processed in batch thanks to scheduling software and a Biostack plate stacker integrated with the EL406.

BlueCatBio Bluewasher/Dispenser

The Bluewasher is a tip-less plate washer. It uses centrifugation to discard liquid from 96- or 384-well plates. The Bluewasher software enables controlling centrifugal rotation speed to either minimize residual volumes of liquids in the well post centrifugation or, conversely, minimizing the disruption of cells monolayers in the well. In addition, the Bluewasher can dispense up to 7 different fluids in 5 microliters increments using a fully automated switching valve. The Bluewasher is used for PFA cell fixation, RNA FISH, and DNA FISH protocols.

Functional Genomic Libraries

For arrayed HCI, the HiTIF offers commercial synthetic RNA oligo libraries of typically 300–1,000 genes per collection, targeting specific cellular pathways or enzymatic families.

For pooled HCI, the HiTIF designed and acquired custom lentiviral CRISPR-KO libraries (typically 500–2,000 genes each) in collaboration with the CCR Genome Modification Core (GMC); on average, these custom pooled libraries cost ~90% less per perturbation than comparably sized arrayed synthetic sgRNA libraries. All libraries are available to collaborators in DNA form.

Arrayed synthetic siRNA and sgRNA libraries

Pooled lentiviral CRISPR-KO sgRNA libraries