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Multicolor Flow Cytometry Best Practices for Optimization

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Multicolor Flow Cytometry Best Practices for Optimization Multicolor Flow Cytometry Best Practices for Optimization BioLegend is ISO 13485:2016 Certified Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.5800 biolegend.com 02-0006-03 World-Class Quality | Superior Customer Support | Outstanding Value Build a Better Multicolor Flow Cytometry Assay Flow cytometric assays of four to six colors are relatively easy to construct and analyze. At this number, you can choose the brightest fluorophores and ensure that they have minimal spectral overlap. However, many researchers prefer to maximize the number of markers in their panel due to limited sample volume or to see the inter-connectivity of many parameters simultaneously. Increasing the number of markers in a multicolor panel requires that a set of guiding principles be followed to ensure that the sensitivity and statistical consistency are maintained between sample and day. As the number of markers in a panel increases, the number of colors with overlapping spectra increases, and so do the complexities of experimentation and analysis. Sensitivity = Signal Brightness − Background (Noise) (MFI+) - (MFI-) Population = Fluorophore Brightness × Resolution Degree of Labeling of the Antibody × Expression Level of the Antigen per Cell Building a balanced panel, titrating and optimizing your antibodies, and using the appropriate controls can all help to minimize aberrations and ensure confidence in your data set. In this brochure, you can learn more about how to get the most out of your multicolor flow cytometry experiment by following these guiding principles. View all of our fluorophore conjugated antibodies at: biolegend.com/flow_cytometry_conjugates Table of Contents Instrument Specifications .............................................................................................................................................................................................................................................3 Balance Antigen Expression vs. Fluor Brightness ............................................................................................................................................................................................4 Fluorophore Considerations .........................................................................................................................................................................................................................................5 Featured Fluorophores .....................................................................................................................................................................................................................................................6 Optimize and Control ....................................................................................................................................................................................................................................................10 Blockers and Buffers ........................................................................................................................................................................................................................................................11 2 Customer Service: 858-768-5800 Instrument Specifications Know your Instrument! Spillover Matrix Reference your instrument’s specifications while constructing When running any flow cytometry experiment, keep in mind a multicolor panel to ensure that you can optimally detect the that high compensation values are not inherently problematic fluorophores that you choose for your experiment. If you don’t as long as you are able to distinguish your positive and negative have this information, contact your flow cytometry facility for populations. Additionally, compensation values can be affected the specifications. Most advanced cytometers have up to 5 lasers by the absolute voltage of each channel. For each experiment, including ultraviolet (355 nm), violet (405 nm), blue (488 nm), use unstained and single stained biological controls to adjust the green (532 nm) or yellow-green (561 nm), and red (633 nm). In voltage for each channel. addition to the lasers on your instrument, there are a limited The spread matrix below was generated using anti-CD4 number of detectors and filter combinations which will dictate antibodies (clone SK3) conjugated to each of the fluorophores how many and which fluorophores you can detect. and run on a Cytek™ Aurora spectral cytometer with 3 lasers Many standard flow cytometers utilize photomultipler (405 nm, 488 nm, 633 nm). On the right, you can see how tubes (PMTs) and specific bandpass filters to detect specific this translates to your experiments as the amount of spillover fluorophores. Alternatively, some newer instruments utilize increases. spectral unmixing for up to 48 fluorescent parameters in a single assay. This is done, in part, by integrating the full spectral signature of fluorophores with very similar spectra, such as Brilliant Violet 421™ and Pacific Blue™. In lieu of using PMTs, these cytometers have Avalanche Photodiode (APD) detectors, which also enables the detection of fluorophores in the near-IR and infrared wavelengths. 106 105 104 acic Blue™ 103 P Channel -103 e™ 700 488 ™ ™ ™ ™ ™ ™ ™ 750 ™ ® 647 lu ™ 594 3 3 4 5 6 P B -10 10 10 10 10 C 510 570 anine5.5 e™ 711 uor® uor uor® 605 650 750 785 421 rC anine7 anine5 c ir y PE zzle y Fl Fl Fl APC AP BV BV BV BV BV BV BV a ci Pe BV a a a C/Fire™ 810 Pa PE/C ex ex ex PE/C AP APC/F Spark NIR™ 685 Spark Blue™ 550 PE/D Al Al Al PerCP/Cy 106 BV421™ Pacic Blue™ 5 105 BV510™ 104 BV570™ yanine BV605™ 103 BV650™ PE/C BV711™ BV750™ -103 BV785™ 3 3 4 5 6 Source Alexa Fluor® 488 -10 10 10 10 10 Spark Blue™ 550 APC PE PE/Dazzle™ 594 106 PE/Cyanine5 5 PerCP 10 PerCP/Cyanine5.5 104 PE/Cyanine7 ire™ 750 APC 103 Alexa Fluor® 647 APC/F Spark NIR™ 685 -103 Alexa Fluor® 700 APC/Fire™ 750 APC/Fire™ 810 -103 103 104 105 106 APC 3 biolegend.com Balance Antigen Expression vs. Fluor Brightness 1. Prior to choosing a particular fluorophore/antibody combination, determine the expression level of the antigen you wish to detect. If the expression level is entirely unknown, reserve the brightest fluorophores for this target to ensure the best chance of adequate detection. 2. Understand how a disease state, exogenous treatment, or stimulation may affect antigen expression. For example, while CD4 is abundant in both mouse tissue and human peripheral blood, it is downregulated upon PMA/Ionomycin treatment. In this case, you can try to use a brighter fluorophore for CD4 or gate on CD3+ CD8- events. Keep in mind that antigen expression may be donor- dependent and change over the course of maturation or within different cellular subsets. Background CD19 CD20 CD22 CD38 B Cells UnstimulatedStimulated 5 106 106 10 105 105 luor® 488 luor® 488 104 104 104 103 103 CD4 Alexa F CD4 Alexa F 103 102 MedQb -102 -102 -103 -102 103 104 105 106 -103 -102 103 104 105 106 2 CD8 APC/Fie CD8 APC/Fie 10 Human PBMCs were isolated and stained without stimulation (left) or stimulated (right) for 6 hours with Cell Activation Cocktail (Cat. No. 423303) and stained with surface antibodies. e r y to ells Naïv ec emor IgM only tural E Plasma C Na Switched M Expression of CD markers changes across different cell types and during maturation. Data provided by CDMaps. 3. Refer to our Brightness Index to help match the potential Brightness Index abundance of the antigen in your assay with an appropriately Fluorophore Cytek™ Aurora Excitation Emission Brightness bright fluorophore. Keep in mind that a bright fluorophore Channel Max (nm) Max (nm) Ranking (1-5) isn’t always the best fluorophore. PE/Cyanine5 B7 488/561 665 5 Brilliant Violet 421™CD8+ T Cells V1 405 420 4 In this table, every fluorophore was conjugated to an anti- Brilliant Violet 605™ V9 405 605 4 CD4 antibody (clones RPA-T4 and SK3) and normal human Brilliant Violet 650™ V11 405 650 4 whole blood was stained. The fluorophore brightness Brilliant Violet 711™ V13 405 711 4 score is a relative indication of fluorescence intensity above Brilliant103 Violet 785™ V15 405 785 4 background and is calculated using the stain index ((MFI PE B4 488/561 575 4 of the positive population - MFI of the negative population)/ Brilliant Violet 750™ V14 405 750 3 2 * Standard Deviation). These values can differ depending PE/Dazzle™MedQb 594 B6 488/561 615 3 102 upon the flow cytometer, instrument filters and settings, laser Alexa Fluor® 647 R1 650 675 3 power, the antibody clone, antigen target, F/P ratios, and the APC R1 650 665 3 buffer conditions, etc. Brilliant Violet 570™ V8 405 570 2 PE/Cyanine7101 B13 488/561 780 2 Pacific Blue™e y y V3 A 405 450 1 Brilliant VioletNaïv 510™ V5 405 510 1 TEMR Alexa Fluor® 488 B2 488 520 1 FITC tor Memor B2 488 520 1 ec Central Memor Spark Blue™E 550 B3 516 548 1 PerCP/Cyanine5.5 B8 488 680 1 Spark NIR™ 685 R2 660 690 1 Alexa Fluor® 700 R4 695 720 1 4 APC/Fire™ 750 R7 650 790 1 Customer Service: 858-768-5800 Fluorophore Considerations When choosing your fluorophores, there are a number of factors Tandem fluorophores are the union of two fluorescent molecules to keep in mind. Ideally, you want to spread your markers across which undergo fluorescence resonance energy transfer (FRET) multiple lasers and choose fluorophores that have minimal upon excitation. A donor fluorophore, such as PE, APC, or Brilliant spectral overlap. As your panel grows, there is a higher likelihood Violet 421™, is excited by the
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