DA-Cell Washer and Plate for Flow Cytometry. Accelerating Life Sciences

Transcription

1 DA-Cell Washer and Plate for Flow Cytometry Accelerating Life Sciences

2 Conventional Centrifuge-based protocol for staining cells Challenges of staining cells for flow cytometry by centrifuge Losing cells non-uniformly more than 30 % Stressing cells Disrupting surface staining Significant user variability Labor intensive Leading to subjective data analysis and diagnosis with possibility of bias 2

3 DA-Cell Replaces Centrifuge step in staining cells Blood Conventional plate Centrifuge for washing Reagents DA-Cell plate DA-Cell washer for washing Centrifuge-less wash offers a number of expected and unexpected benefits Flow cytometer: read tubes, a conventional plate, or DA-Cell plate 3

4 Key Features of DA-Cell System in staining cells DA-Cell HT processes up to 96 samples in parallel in an SBS standard format A plate can be used partially over a period of 20 days One well of a DA-Cell plate can hold up to 70 ul and wash from single cell to ~5 million cells DA-Cell HT washer is a compact plug-and-play with a fully automated and optimized wash program Typical wash process entails dispensing and aspiration of ~60 ul 5 7 times for min duration 4

5 Key Benefits of DA-Cell technology in staining cells Retaining more than 99 % cells even for intracellular staining Improving cell viability by centrifuge-less washing Producing accurate data consistently and reproducibly independent of an operator Significantly easier gating and segregation of cell populations Washing completed within 2-4 minutes skipping centrifuge step Reliable segregation of cell populations even with a higher number of colors Complete cell retention and no stress on cells indisputably lead to more accurate and reproducible data for any given assay 5

6 DA-Cell Washer equipped with 192 nozzles, 2 nozzles per droplet of DA-Cell Plate Before washing: two drops with ink and two drops with green PS beads of 6 mm After ~4 min washing: ink washed away and beads retained DA-Cell needs just 2-4 minute for the wash equivalent to 2-4x centrifuge wash of a microtiter plate 6

7 Magic of DA-Cell: Gravity force and Laminar flow Cell centrifugation washing method Repeat 2-4 times Mixing of cells with fluorescent marker Incubation of cells for 20 min Packing of cells via centrifugation Supernatant removal Washing buffer addition & mixing Manual min hands on time DA-Cell washing method Mixing of cells with fluorescent marker Incubation of cells for 20 min and natural settlement of cells Laminar flow based washing with minimal nonspecific binding One step of preprogrammed wash Automated 3-5 min hands on time Laminar flow washing provides as effective washing as centrifuge if not better 7

8 DA-Cell Leads to Better cell resolution and Easier gating Speculation of two primary factors No squeeze and aggregation of cells from centrifuge leading to clean, uncompromised binding of antibodies onto a cell surface More extensive washing of bigger dilution factor The comparison of flow plots from DA-Cell, Microtiter plate and FACS tube indicates both factors MATTER while the absence of centrifuge more dominant Droplet-based DA-Cell method Existing centrifuge method Washing Uniform laminar flow leading to minimal sample loss and complete wash Capillary action leading to turbulent flow and cell loss 8

9 Increased cell retention to more than 99 % by slightly longer incubation Wash Incubate 20 min >95% settled Retention of >95% Vortex 30 sec Wash Incubate 30 min >99% settled Retention of >99% The principle of cell retention works equally well for those fixed and permeabilized cells as well as live cells 9

10 Minimal loss of cells during washing An example of cell loss during a typical washing of fixed and permeabilized cells Dispense 1 million permeabilized and fixed cells in 50 ml Perform DA Cell washing by the following manner Aspirate 32 ml Dispense 62 ml followed by aspiration of 62 ml Repeat the dispensing and aspiration as needed, typically 5-7 times Collect the aspirated solution in a separate vial, spin down, and count the number of cells A table below shows an example of cell loss test performed for 1 million perm/fix cells added onto a well of a DA-Cell plate: one wash step loses 0.05~0.6% cells 1 st Aspiration 2 nd Aspiration 3 rd Aspiration 4 th Aspiration 5 th Aspiration Total loss Run 1 ~5,000 ~500 ~100 ~100 ~100 ~5,800 (0.6%) Run 2 ~5,000 ~100 ~100 ~100 ~100 ~5,400 (0.5%) Run 3 ~100 ~100 ~100 ~100 ~100 ~500 (0.05%) 10

11 Typical cell staining protocol by DA-Cell same as Centrifuge Cryopreserved cells were thawed and counted Cell concentration were adjusted to 50ul/Million PBMCs Cells were aliquoted by 1 million per well A mastermix of antibodies by total volume of ul was added followed by quick mixing and min incubation Cells were washed accordingly a microtiter plate by centrifuge and a DA-Cell plate by DA-Cell washer Cells were collected from a plate and transferred to a tube for data acquisition by flow cytometer 11

12 Example of comparing DA-cell and Centrifuge method with PMBCs Type of Cells to be enumerated for two donors PBMCs inkt MAIT GDT VD2 CD4 CD8 CD4/8 Naïve/Memory/Senescent Phenotype NK Cells and Subsets B Cells (Naïve, Memory) HSC Monocytes and Subsets pdcs Basophils 21 Color Stain Marker Flourophore Clone Company CD45 FITC H130 Biolegend Va24 J18 PerCP Cy5.5 6B11 ebioscience CD34 PE 563 BD CD19 PE Cy7 HIB19 BL CD14 PE Texas Red HCD14 BL IgD BV785 BL VD2 BV711 B6 BL CD27 BV650 O323 BL Va7.2 BV605 3C10 BL CD4 BV570 SK3 BL CD161 BV510 HP 3G10 BL CD57 BV421 HCD57 BL CD3 BUV395 UCHT1 BD L/D BUV496 Lifetech CD56 BUV563 NCAM16.2 BD CD16 BUV737 3G8 BD CD8 BUV805 SK1 BD HLA-DR AF700 BL GDT APC 11F2 MACS CD45RA APC Fire750 H100 BL CD123 PE Cy5 6H6 BL BSB 12

13 More Effective and Accurate Segregation by DA-Cell: Example of 16-color assay HSC DA-Cell Plate inkt HSC MTP (Microtiter plate) inkt GDT MAIT GDT MAIT 13

14 More Effective and Accurate Segregation by DA-Cell: Example of 21-color assay T cells DA-Cell Plate inkt T cells MTP (Microtiter plate) inkt IGD MAIT IGD MAIT 14

15 DA-Cell Enables running a high number of color DA-Cell Plate MTP 6-stain 11-stain 21-stain * Red/Blue colors in heatmap is desirable. White color means unidentifiable due to unclear signal Heatmap shows the outcome of unbiased, automated gating and clustering DA-Cell allows a same quality of clustering and annotation as the number of color increases DA-Cell produces data of clear resolution even at 21-color assay In contrast, MTP fails in resolving populations as the number of color increases DA-Cell enables running a high number of color with minimal deterioration of data quality In collaboration with Anis Larbi group at Singapore Immunology Network (SIgN), A*STAR 15

16 Better cell resolution and Easier gating by DA-Cell CD34 CD3 DA-Cell FACS Tube MTP Effectiveness of rinsing is at DA- Cell FACS Tube x2 > MTP x2 at 8,000x and 500x dilution, respectively DA-Cell shows superior cell resolution over the other conventional methods suggesting pelleting of cells responsible for poor resolution GDT In collaboration with Anis Larbi group at Singapore Immunology Network (SIgN), A*STAR 16

17 Same Workflow with less hands-on time and better consistency: example of intracellular staining assay Microtiter Plate (MTP) or Tube 1M cells + master mix 100 ul Incuba tion Manual wash 2x Addition of perm/fix buffer 100 ul Incuba tion Manual wash 2x Addition of master mix buffer 100 ul Incuba tion Manual wash 2x Additi on Read DA-Bead Plate Flow 1M cells + master mix 50 ul Incuba tion Automated wash Addition of perm/fix buffer 50 ul Incuba tion Automated wash Addition of master mix buffer 50 ul Incuba tion Automated wash Additi on Less reagents Less reagents Less reagents 17

18 Extremely Easy Operation and Maintenance DA-Cell installation and operation Compact size of 11" x 12" x 12" (WDH) of ~20 lbs Clean and prime with 70% EtOH, DI water, and then FACS wash buffer Input the size of a drop on a DA-Cell plate default 50 ul Input the number of dispensing/aspiration cycle default 7 A liquid handling head can be replaced within a minute by a user 18

19 DA-Cell Washer and Plate Critical for Accurate Data Complete cell retention and no stress on cells indisputably lead to more accurate and reproducible data Accurate data and analysis achieve long term saving of resources Improved productivity, happier workforce and saving of reagents offer quick return on investment Curiox offers various purchasing options For free on-site demo and trial, please contact your account manager or 19