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Healthy CellsBroad CapabilitiesBetter ScienceGentle, customizable benchtop microfluidic cell sorting. 2022 NanoCellect Biomedical, Inc. All rights reserved.1

High cell integrity without compromiseThe WOLF was originally created by a team of scientists and engineers who wanted to solve a classicchallenge in biological research: how to effectively and easily sort high-quality cells. Now, as sciencecontinues to move forward into increasingly complex realms, the WOLF is moving forward with it. TheWOLF G2 instrument has significantly expanded the capabilities of gentle benchtop microfluidic cell sortingwith two lasers and up to nine colors, while maintaining simple workflows for either bulk sorting or singlecell dispensing. Single-cell sorting can be completed in 96- or 384-well plates when using the WOLF G2in conjunction with the N1 Single-Cell Dispenser. This flexibility in performance, along with the additionalabilities of the second laser, makes it ideal for use in many different research fields and application areaslike single-cell genomics, cell line development, gene editing, antibody discovery, immunology, infectiousdisease, basic research, and more.WOLF G2 Cell SorterHealthy CellsContaminant- and Biohazard-FreeAt 2 psi, the WOLF G2 are gentler than anyconventional cell sorters, enabling healthiercells post-sort, especially for engineeredlines, primary cells, and stem cells.Disposable, aerosol-free microfluidiccartridge isolates the sample from theenvironment, while protecting the userfrom exposure.High Sensitivity and ResolutionCompactAll laser configurations afford 250 MESFsensitivity, along with forward and backscatter, providing as low as 1 µm resolution.At under 2 cubic feet, NanoCellect’sbenchmark for access and performance allowsevery lab for the flexibility to do analysis andsorting into tubes or 96- and 384-well plates.Simple and at your BenchIntuitive software, fixed optics, nofluidics cart and less than one minuteclean-up time.Expanding the WOLF’s CapabilitiesWith two lasers and up to ninefluorescent channels, the WOLF G2 alignswith a broad set of research applicationsand experiments. Three different laserconfigurations allow options specific toyour needs. 2022 NanoCellect Biomedical, Inc. All rights reserved.1

ApplicationsGene EditingCNS PatientiPSCs CRISPR Gentle microfluidic cell sorting with theWOLF uses pressures of below 2 psi.The combined power of the WOLF andthe N1 single-cell dispenser eliminatesthe main challenges typically facingpluripotent stem cell processing.Sterile, Gentile SortingSingle-Cell Plating Without decompression shock and shearstress, single-cell deposition results inhigh monoclonal outgrowth.Karyotype This improved workflow will allow forhigher throughput pluripotent stem cellresearch, which is valuable for bothbasic and clinical research.Infectious DiseaseViral Particlesor AntigensViral Culture The WOLF G2 microfluidic cell sorter is aerosol-free andcompact enough to fit into standard biosafety hoods, making itideal for infectious disease research.Biohazard Antigenand B cell SortingTherapeuticAntibody Production The WOLF G2’s disposable cartridges also avoid crosscontamination and create a safe and effective workflow forthe kind of speed and efficiency required to combat infectiousdisease threats.Immunology and Antibody DiscoverySplenocyteExtractionSingle-Cell SortingAntigen and B Cell The WOLF G2 Cell Sorter provides a simple cell sorting solutionthat gently sorts high antibody-producing clones and dispensessingle cells for optimal clone outgrowth.2VDVLLHeavy constantLight constantRT-PCR and CloningAntibody Scale-Upand Production With two lasers and up to nine fluorescent channels, the WOLFG2 provides the most comprehensive benchtop cell sortingsolution. 2022 NanoCellect Biomedical, Inc. All rights reserved.

Cell Line DevelopmentDAY 14DAY 5DAY 0SequenceTransfect and CultureSterile Plate SeedingPlate Viable Single CellsImage Single CellsScale-UpProduction The WOLF G2 Cell Sorter protects cell viability after sorting andimproves sorting efficiency by increasing the number of viableclones growing per plate. When paired with the N1 Single-Cell Dispenser, the WOLF canselect viable single cells and plate them into 96- or 384-wellplates. Contaminant- and biohazard–free, the WOLF cell sorter’s rapidlyexchangeable cartridge and tubing set eliminates carryoverbetween samples and allows for quick and easy cleanup. Low pressure microfluidics greatly improves cell integrity overconventional droplet sorters, resulting in high-outgrowth andhigh-titer monoclonal lines.GenomicsBloodFilter The WOLF G2’s gentle microfluidicsystem eliminates unintentionalsequencing and analysis of dyingor off-target cells or debris whilemaintaining cell integrity andavoiding stressing cells and affectingdownstream data.Sample Prep Researchers can sort cells with awide range of genome sizes, includinghuman, plant, and animal cells ormicroorganisms like yeast and bacteria,providing insight on population geneticsor disease. 2022 NanoCellect Biomedical, Inc. All rights reserved.Single-CellNGS The WOLF G2 also allows labs to becompletely flexible when it comes tosequencing research and eliminatescarryover with a 100% disposablefluidic cartridge.3

Microfluidic Cartridge TechnologyThe WOLF Cell Sorter uses patented, microfluidic-based sorting with robust laser-excitation and sensitive PMT detectors to isolatemammalian cells, microbes, plant cells and more. A gentle and precise piezoelectric actuator directs cells into collection channels andallows analysis and sorting in a disposable format. This eliminates sample-to-sample contamination and biohazard exposure or cleanup. Unique to NanoCellect are our disposable cartridges that allowfor bulk sorting or single-cell sorting. The sorting cartridges use a piezoacoustic actuator that gentlydirects cells into collection channels; an embedded cell sortingverification system gives instant feedback of sorting accuracy. This technology allows the WOLF G2 to sort up to 200 cells persecond with high accuracy and effective recovery. Sort two selected cell populations with bulk sorting while theremainder of cells collects in a third channel. Deposit 1 to 100 cells per well in a 96- or 384 well plate usinga single-cell sorting cartridge along with the N1 Single-CellDispenser accessory.Magnified Sorting JunctionActuatorKey BenefitsSterileNo aerosolsCartridges are individually wrapped in sterilepackaging to avoid cross-contamination.No dangerous aerosols are produced duringsorting of hazardous samples.DisposableEasy clean-upAnything that the sample or sheath fluidtouches is disposable.Clean-up in one minute and simply discardthe cartridge.Gentle SortingA gentle sorting mechanism results in improvedviability of cells and higher outgrowth.4 2022 NanoCellect Biomedical, Inc. All rights reserved.

WOLF G2 Cell Sorting5-Color Immune Cell Sorting:To verify sorting performance, CD4 and CD8 T Cells were sorted from BioLegend’s PBMC Veri-Cells. They were gated from theCD3 CD19-CD45 lymphocyte population.LymphocytesSingletsCD45 CD3 CD19-CD4 and CD8 5-Color Sort Performance:Transfected Cell Lines:CD4 and CD8 T Cell populations were evaluated based ongates. CD4 T cells were enriched to 92.0% from a 26.5%target population. CD8 T cells were enriched to 91.7% froma 13.9% target population.Four HEK293T cell lines were mixed to mimic a GFP RFP dual-expressor transfection. To verify sorting performance,the GFP RFP cells were sorted from the rest to result in90.5% post-sort purity.Pre-SortCD4 Post-SortHEK293T CellsSingletsMock-Transfection MixPost-Sort: GFP-RFP CellsCD8 Post-Sort 2022 NanoCellect Biomedical, Inc. All rights reserved.5

Monoclonal HEK OutgrowthThe same 25% GFP RFP dual-expressor cell mixwas also sorted for single cells into 96-well platesand incubated for 14 days. As a rigorous limitingdilution comparison, 100% GFP RFP dualexpressor cells were dispensed at 1 cell/well. TheG2 plate still yielded more than a 2-fold increase intargeted monoclonal colonies.Limiting Dilution: 1 cell/well from 100%GFP RFP HEK293T cell mixLittle to no fluorescenceN1 Single Cell DispenserDesigned to sort and dispense into 96- and 384well plates, the N1 provides higher rates of singletdetection compared to cell printers or limiting dilution.Users can perform simple, label free dispensing oradvanced multicolor panel single-cell dispensing.Plate sorting specificationsTime to plate (96 wells): 3 - 8 minutesTime to plate (384 wells): 32 minutesDroplet volume: 3 - 10 µLSample plate options: 96 or 384 wells(flat bottom, V-bottom, U-bottom, PCR)WOLFViewer SoftwareThe WOLFViewer software has an intuitive workflowmenu that walks users through their experimentalprocess, and is designed for both novice and expertusers. New users can be performing their first sorts inabout 20 minutes and can be on their own on day one.Plate sorting specificationsSystem power up: 3 minutesSystem setup and calibration: 20 minutesSystem shutdown: 3 minutesAdditional features: Auto-alignment, intuitivecompensation, advanced coloring and gatingoptions, FSC files compatible with FlowJo6 2022 NanoCellect Biomedical, Inc. All rights reserved.G2 Single-cell Dispense: 1 cell/well from25% GFP RFP HEK293T cell mixRFP only fluorescenceGFP RFP fluorescence

WOLF G2 Configuration GuideThe WOLF G2’s three possible laser configurations significantly enhance the number of fluorescent markers that may be utilized.WOLF G2 405/488WOLF G2 405 nmExcitation SourceEmission Detection: Filters405 nm, 55 mWDPSS laser 450/50 (DAPI, BV421*)525/50 (BV510*)575/40 (BV570*)620/50 (BV605*)706/95 (BV711*)WOLF G2 488 nmExcitation SourceEmission Detection: Filters488 nm, 55 mWDPSS laser 525/50 (FITC, GFP)575/40 (PE)620/50 (PI)706/95 (PE-Cy5.5 )450/5010080WOLF G2 488/561Excitation SourceEmission Detection: Filters488 nm, 55 mWDPSS laser 525/50 (FITC, GFP)580/25 (PE)620/50 (PI)706/95 (PE-Cy5.5 )760LP (PE-Cy7 )WOLF G2 561 nmExcitation Source561 nm, 55 mWDPSS laserEmission Detection: Bandpass Filters 580/25 (PE)620/50 (PI, mCherry)706/95 (7-AAD, PE-Cy5.5 )760LP (PE-Cy7 )575/40Excitation SourceWOLF G2 488/637488 nm, 55 mWDPSS laserEmission Detection: Bandpass Filters 525/50 (FITC, GFP)575/40 (PE)609/34 (PI)706/95 (PE-Cy5.5 )760LP (PE-Cy7 )WOLF G2 637 nmExcitation Source637 nm, 55 mWDPSS laserEmission Detection: Bandpass Filters 706/95 (APC) 760LP (APC-Cy7 )706/95405 nm40200DAPI525/5010080BV510BV570 BV605575/50BV711620/50706/95488 706/95760/LP488 5PE-Cy7760/LP561 nm6040200WOLF G2 488 nm620/50600WOLF G2 488 nm525/50PE525/5010080PI575/40 609/34PE-Cy5.5706/95PE-Cy7760/LP488 637 nm40200 2022 NanoCellect Biomedical, Inc. All rights reserved.APCAPC-Cy77