cytoflex compensation beads

Platelet Flow Cytometry: Instrument Setup, Controls, and Panel Correspondence to The mean and median sizes are also included in the table below, the SEM. Some of these factors may have no substantial benefit on their own, but they combine to great effect. The single biggest innovation that enables such dramatic performance improvement on the CytoFLEX is the use of small-area APDs for signal detection. Plasma was prepared from fresh human blood by a combination of centrifugation and filtration. Only in case of the Fusion 2 another higher laser power setup was used to test the effect of laser power on spread. PDF CytoFLEX - Beckman Slider with three articles shown per slide. 4B45. conducted all flow-cytometry experiments. PubMed Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Incubate 15-30 minutes at room temperature. The sensitivity of SSC and VSSC on the CytoFLEX can be seen in Supplementary Fig. Nanomedicine 14(3), 801810 (2018). prepared the samples and reagents. One of the major problems with the CytExpert 1.2 was the problems that existed with compensation when using a template which was supposedly corrected in this version. If a new compensation is needed, generate as follows: PDF CytoFLEX Daily Startup, QC and Shutdown - Memorial Sloan Kettering For the CytoFLEX LX system, 710/50 bandpass filters on the blue and the red lasers were used . The details and rules of rectifying this unwanted signal in secondary detectors, compensation, can be found extensively explained by others4,5,6,7,8. For this reason, the factor of rSD required to calculate the 99th percentile for each detector is highly variable. CAS The constant nature of P in these terms allows for direct comparison of SQI values between instruments with different dynamic ranges. However, the voltage changes do affect the ISS values considerably. G.C.B., Y.Q.C., S.G. and M.-A.L. 22(6), 774775 (1983). This ensures that you do not run out of sheath fluid during an experiment. CytoFLEX Flow Cytometer by Beckman Coulter Life Sciences ). CD61+ platelets and platelet EVs in whole blood are shown in Fig. Cytometry 10, 426432 (1989). Prepare the QC beads: Add 1mL of deionized water to 12 x 75mm sample tube. In the discussion, we elaborate on the conditions that must be met. 83(3), 306315 (2013). PDF Creating Compensation File in CytExpert - pedsresearch.org Flow Cytometry Facility, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC, 27858, USA, Flow Cytometry Facility, The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands, The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands, Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC, 27858, USA, You can also search for this author in J. Virol. The SQI values remained the same for both detectors in all cases, but the ISS values decreased considerably. While these methods may provide insights into EV biology, they ultimately obscure individual particle characteristics and, thus, the ability to properly analyze EV populations and subpopulations. 22, 1.14.1-1.14 (2002). Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages. (B) The detection of 98.6293nm Si particles by VSSC. Flow cytometer. Google Scholar. Bennet, H. S. & Rosasco, G. J. Resonances in the efficiency factors for absorption: Mie scattering theory. VSSC gain=400; VSSC-H threshold=3000. . EMBO J. 4EH). J. Softw. In Supplementary Fig. The CD61+ EVs prepared using this method were roughly 50% CD9 double positive, while they were predominantly CD63 and CD81 negative (Fig. Sci. However, this example shows that it is possible to use SQI values for the purpose of comparing instrument performance with regards to spread for a given set of fluorochromes. We did this change to introduce more spread in the APC and PerCP-eFluor 710 detectors. wrote the manuscript. The optimal threshold setting for the CytoFLEX S N-V-B-R was determined empirically using 81nm PS nanoparticles at their optimal dilution. As recommended in Ref.13, cells were 1st stained with TCR antibody for 15min then stained with the master mix of the rest of the antibodies for another 15min. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Diluted beads can be stored at 2-8C in dark for up to 5 days. Cytometry A. 117, 5.4.1-5.4.38 (2017). Ann. Move the positive and negative gates to enclose the corresponding peaks. Appl. & Gershon, P. D. Structure of intracellular mature vaccinia virus visualized by in situ atomic force microscopy. Opt. Sci Rep 9, 16039 (2019). All the methods were carried out in accordance with relevant guidelines and regulations. is the CEO and V.A.T. We thank Dr. Alexandra Terry for providing the cells. 3: HAdV-5 (A), HIV-1 (B), MLV (C), and HSV-1 (D). The filtered PPP was then passed through Izon qEVsingle SEC columns with a 70nm pore cutoff in order to eliminate the abundance of proteins and lipoproteins present below the size cutoff for the columns, while further narrowing the size distributions in the different column fractions. It is a mathematic procedure based on a fundamental constant proportional relationship between the two signals, Figure 1. However, care should be taken when using this method. If a new compensation is needed, generate as follows: Prepare all necessary unstained and single color controls using compensation beads or cells. Using VSSC versus SSC for small-particle detection has an added benefit that the shorter-wavelength light actually increases the amount of light scattered by small particles due to increasing the relative refractive index (RI)19. 98.6nm Si particles with a RI of 1.44 can be fully resolved by VSSC. Each of these are known to have biases for particular small-particle populations based on their densities, sizes, surface charges, or other properties, and each result in variable levels of residual protein and lipoprotein contamination5,6. In total, 7.5% of the CD61 EVs from Donor 2 were triple positive. Finally, we are certain that the voltage and dynamic range independent nature of SQI values will be instrumental in preventing manipulation of detector gain settings to minimize spread, rather than to use settings that provide the best separation and sensitivity. DB like to thank Lyndsay Richard for all her help. https://doi.org/10.1038/s41598-021-99831-7, DOI: https://doi.org/10.1038/s41598-021-99831-7. J. Quant. The cells were then filtered gently and centrifuged at 500g for 10min. CAS The SQI values were the same in both voltage sets, but the ISS values were quite different (Table 3). Shapiro, H. M. Practical Flow Cytometry 4th edn. Chen, Y. Q. Useful tools/methods have been developed to aid investigators in this process. Flow Cytometer Calibration and Size Reference Beads The enhanced sensitivity of the CytoFLEX vs. other instruments, even when using diode lasers with less than half of the power output of traditional lasers, is again due to the enhanced optics collecting approximately 110 of orthogonally scattered light, the WDMs minimizing signal losses, and the small-area APDs having a high quantum yield with minimal electronic noise. However, because the intensity values in the primary detector dont influence the SQI values, this is not necessary in our method. Clone matched (SK3) Alexa Fluor 647 conjugated CD4 antibody (cat no 344635), and anti-human Alexa Fluor 647 conjugated CD45RA antibody, clone HI100 (cat no 304153) were obtained from BioLegend. A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles, \({\rm{SD}}=\sqrt{\frac{{\sum }_{i=1}^{N}{({x}_{i}-\bar{x})}^{2}}{N-1}}\), \({\rm{C}}{\rm{V}}({\rm{ \% }})=(\frac{SD}{Mean})\times 100{\rm{ \% }}\), $$Theoretical\,Intensity=(\frac{Actual\,VSSC\,Intensity\,}{Reference\,Scatter\,Efficiency\,})\times Theoretical\,Scatter\,Efficency.$$, https://doi.org/10.1038/s41598-019-52366-4. Internet Explorer). conceived, performed, and analyzed experiments. However, the ISS values changed noticeably. Article Our control beads offer: Confidence help ensure the reliability of optimal daily instrument performance Each sample was read in triplicate or more, and the population means were calculated by averaging the median or mean intensity measurements from each individual data point. 2032, 129 (2019). At an output power of 140mW, the Red laser resulted in lower spillover spread from APC and APC-R700 compared to lower output power of 100mW (Table 2). and T.M.R. Nguyen, R., Perfetto, S., Mahnke, Y. D., Chattopadhyay, P. & Roederer, M. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Sahir, F., Mateo, J. M., Steinhoff, M. & Siveen, K. S. Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry. XB50=50th percentile of compensated positive population of fluorochrome X in detector B. XB99=99th percentile of compensated positive population of fluorochrome X in detector B. UnB50=50th percentile of compensated unstained population of in detector B. YStB50=50th percentile of compensated stained population of fluorochrome Y in detector B. NFYB=Normalization factor of fluorochrome Y in detector B (Normalization Factor, in Fig. CytExpert software separates compensation and sample acquisition into separate experiments designated .xitc and .xit, respectively. 121, 920922 (2017). 50L of plasma or EV samples were aliquotted into different 1275mm tubes, and an aliquot of the antibody mixture was added to each sample. Using this approach, we narrowed the size range down to roughly 70200nm for comparison between DLS and flow cytometry. When analyzing for specific populations (Supplementary Fig. The samples were stained for another 30min in dark at 4C. Finally, the matrix of calculated VSSC intensities was plotted versus size, with the different curves representing contours of RI equivalencies (Fig. The best way to avoid this problem is to find a combination of bright fluorochromes with dimly expressing antigens. CE: Products intended for in vitro diagnostic use and conforming to European Directive (98/79/EC). Spectrosc. 1 106 cells were re-suspended in 100l of staining buffer. 8, 25962607 (2010). Further details on the RI calculations for each donor can be found in Supplementary Figs1114. By submitting a comment you agree to abide by our Terms and Community Guidelines. Next, a matrix of Mie-theory scatter efficiencies is prepared for different RIs using the size of interest, and this matrix is converted to scaled instrument intensities as above, using the approximate scatter intensity previously calculated as the reference intensity. 15(22), 68306840 (2009). To label with antibodies, the antibodies were first mixed in their appropriate concentrations in a master mix. The CytoFLEX can detect as small as 60nm PS nanoparticles, and can resolve as small as 70nm. This enhanced functionality is perhaps uniquely capable of helping scientists and clinicians to achieve their goal of developing EV-based liquid biopsies for hard-to-detect diseases, such as tissue-resident cancers, minimal residual disease, and even neurological disorders. New technologies for analysis of extracellular vesicles. 7 X 106 Cells were thawed in a 37C water bath. Select the channels requiring compensation and then select, Load the corresponding unstained or single color sample fluorospheres or cells and select. Article PubMed Central PubMedGoogle Scholar. This level of sensitivity can greatly benefit exosome and EV research, as most flow cytometers have been suggested to be unable to detect EVs smaller than 300nm in diameter11. Sitemap. Google Scholar. Sci Rep 11, 20553 (2021). Proc. The Spillover Spreading Matrix (SSM) can be used to estimate the spread, but the outcome is dependent on detector sensitivity. In a separate experiment single stained beads were run for a series of PMT voltages. 5, 78, https://doi.org/10.3389/fcell.2017.00078 (2017). 64, 676705 (2012). CD4 kits consist of many different tandem dyes. Article Voltages in group one was set to place the positive population at~110,000 MFI. SQI again remain same but ISS increased with voltage. Run the CytoFLEX System Startup Program. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The lower detection limit for additional particles with a RI of 1.47 would be approximately 81nm at the threshold and 86nm at the level of 60nm PS beads (Supplementary Fig. Small-area APDs have a higher quantum yield, increased linear dynamic range, and minimal dark-current electronic noise13,14,15. b . Range was calculated for all spillover signals. PDF CytoFlex Quick Guide - Beth Israel Deaconess Medical Center Moreover, experimental characterization of the resulting samples generally consists of bulk methods, including western blots, bead-based sandwich assays, genomic assays, dynamic light scattering (DLS) and nanoparticle tracking analysis4,7,8. For Quanteon and CytoFLEX, positive signals were set to approximately 250,000 and 1,000,000 MFI. This is reciprocally demonstrated by analyzing the CD81 expression on CD9+ CD61+ EVs, and the CD9 expression on CD81+ CD61+ EVS. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Google Scholar. Front. 14)Close soware. If necessary, adjust the scatter gate so that it encloses the desired population. Protoc. Formalin-inactivated HIV-1 (HV-H-Zero) and MLV (MV-M-Zero) were obtained from ViroFlow Technologies, Inc. (Ottawa, Canada). G.C.B. The output of the detectors is displayed using a scale, which can vary depending on the vendor and model. The spread remained the same as was evident by the unchanged rSD, but the ISS values changed because the value of F changed. Most noise of this nature resides in the scatter range below 100nm PS beads, so this may not be a notable issue for less sensitive flow cytometers. J. Virol. Article For PS nanoparticles, we found that the CytoFLEX could detect PS beads as small as 60nm, though these beads did not achieve baseline separation (Fig. S4) demonstrate that SQI is a platform independent method that will aid in better panel design. We then tested a variety of small and low-scatter particles, including both synthetic beads and viruses, in order to better assess the resolution limit for VSSC on the CytoFLEX. A gain and dynamic range independent index to quantify spillover spread to aid panel design in flow cytometry. no. In (D) only voltage of V677 was lowered which resulted in a huge increase of ISS but SQI remain same. This needs to be the same between repeats to ensure the difference in number of photons (P) between the positive and negative populations remain equal between runs. We like to thank Dr. Morten Nrgaard Andersen for his crucial critical comments. 2. The 68.6 (NS-0070A) and 98.6nm (NS-0100A) NIST-traceable Nanosilica beads were from MSP Corporation (Shoreview, MN). Online Terms of Use We presented a special scenario where the SQI value of one fluorochrome was calculated for two different fluorochromes, used separately in the secondary/recipient detector (Supplementary Table S1). Vesicles 3, 25361, https://doi.org/10.3402/jev.v3.25361 (2014). Comparing percent of scale units occupied with SQI will provide the user with a better understanding of spread between different types of machines (Table 1) and between multiple machines of the same type (Table 2). Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. CAS Saturated single stains of six different fluorochromes (BB700, BV605, BV711, PE-CF594, PE-Cy5 and APC-R700) were prepared as described above. However, fluorochrome stability, of tandem dyes specifically, is of major concern since tandem dye replacement or time dependent degradation will affect the outcome. (YStB50 UnB50)=The difference between the positive and negative populations (scale units) (Difference 2, D in Fig. Hale, G. M. & Querry, M. R. Optical constants of water in the 200-nm to 200-m wavelength region. (AD) VSSC-H population statistics for (A) HAdV-5, (B) HIV-1, (C) MLV, and (D) HSV-1. To resolve this problem, we perform a daily clean using at least 6 wells of the cleanse solution, followed by 12 wells of water after each QC run. This setup procedure is required under the following situations: You are running the LEGENDplexTM kit for the first time. Chen, Y. Q. The experimenter can choose which one to execute first. (D) CD61+ EVs in PPP filtered through a 0.2m filter, with platelets eliminated. Biol. Therefore, SQI values can be directly compared between any instruments. Front. Indeed, solving Mie-theory equations to determine specific particle characteristics typically requires a variety of complex variables pertaining to the internal engineering of the flow cytometer, which may not be readily available39,40,45,51,52. The data from (AC) were overlaid on the plot to verify the accuracy of the scaling. The quantity of light scatter produced by particles depends directly on their RI, which complicates the ability to determine the size of particles based on reference standards with a different RI. A Compensation C B Creating New Experiments D E Remote Support A. (B) Overlay of the average VSSC intensity vs. DLS size measurements for each EV fraction on the scaled Mie-theory RI curves prepared in Fig. The experimenter can choose which one to execute first. PDF CytoFLEX - media.beckman.com Correspondence to 1A). Sci. CytoFLEX Flow Cytometer Quick Start Guide Adjusting Settings and Compensation Compensation 1. G.C.B. A first statistical model, using EM algorithm, is performed on the absolute value of the intensities measured by the primary detector corresponding to fluorochrome X. Willms, E., Cabaas, C., Mger, I., Wood, M. J. Extracellular vesicles (EVs) are small, naturally occurring cell fragments that range in size between 301000nm. Antibody LIVE/DEAD fixable dead cell stain BrightComp eBeads (for fluorescent proteins) UltraComp eBeads Plus compensation beads Are you using more polymer dyes from the violet and UV lasers? Maximal excitation makes P practically a constant. PDF Guide for Using BD Cytometric Bead Array (CBA) Flex Sets with the BD Appl. Vaccinia Virus (VV) is a large, ellipsoidal DNA virus, with highly variable size characteristics in literature. provided critical review of the manuscript. Kits on the CytoFLEX flow cytometer utilizing the innovative CytoFLEX compensation library. The lower number of double positive events registered for the PE Cy5 compare to BV421 because of the need to account the higher spreading error coming from the PE Cy5. Fusion 1, 2 and 3 were used with identical optical configuration and laser output power unless stated otherwise. 2B). Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. Google Scholar. SQI values are independent of detector type or dynamic range. Sizing Viruses by Flow Cytometry. These reagents are labeled "Analyte Specific Reagents. Whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus. Floderer, C. et al. This calibration is a tedious process. This number will stay practically constant when saturatedly stained samples will be maximally excited. Cytometry 45(3), 194205 (2001). Import the compensation and gains into the New Experiment. In conventional flow cytometers, each detector should ideally receive photons from one specific fluorochrome as a labeled cell travels through the laser interrogation point. For example, laser output powers of the Quanteon and the Symphony were matched, but there was no option to alter the laser power for the CytoFLEX. J. Extracell. Briefly, the 405/10 VSSC filter was moved to the V450 channel in the WDM, while the V450 and V525 channels were each shifted (eliminating the V610 channel), and the detector configuration was modified in the CytExpert software to assign the VSSC channel within the WDM. Detector B is the recipient detector where spread from fluorochrome X is being registered. Acad. Spreads from different machines independent of make/model/dynamic range or vendor can be compared directly using SQI. Finally, we performed size-exclusion chromatography (SEC), using Izon columns with a 70nm pore size, to reduce proteins and lipoproteins. Immunol. Furthermore, the filter configuration (characteristics and positioning) between the instruments was also not the same (Supplementary Table S2). Approximately 8.7% of the CD61+ EVs from Donor 2 were CD81 double positive, including 7.5% that were CD9, CD81 and CD61 triple positive (Fig. All methods were performed in accordance with the relevant guidelines and regulations. PubMed Central Traditional flow cytometers generally collect only 3060 of side-scatter and fluorescent light, typically using focusing lenses. Shao, H. et al. 0.25g of antibody was added to all samples (at saturation). 352054). 4C). Internet Explorer). Varga, Z. et al.
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