Transcript Slide 1
What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry IGC – April 03, 2013 Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34 Outline • Know Your Instrument • Optical Layout (lasers and filters) • Choosing the right fluorochromes • • • • Staining Index Spillover Compensation Color Specificities and Tandem Dyes • Rules for Multicolor Analysis Know Your Instrument Know Your Instrument Reagent Selection starts with Instrument Configuration Analyzers • Lasers • Detectors and respective filters FACScan FACSCalibur CyAn ADP Cell Sorters HyperCyt FACSAria MoFlo LSR Fortessa Know Your Instrument (FACScan) FACScan Optical Configuration Typical Fluorochromes 488 585/42 FL1 FL2 650LP GFP FITC Alexa488 CFSE FL3 488 nm 530/30 400 450 500 550 600 650 700 750 800 PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 Know Your Instrument (FACSCalibur) FACSCalibur Optical Configuration Typical Fluorochromes 488 585/42 670LP FL1 FL2 FL3 GFP FITC Alexa488 CFSE 488 nm 530/30 400 450 500 550 600 650 700 750 PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 800 633 661/16 FL4 633 nm APC Cy5 Alexa647 400 450 500 550 600 650 700 750 800 Know Your Instrument (CyAn ADP) CyAn ADP Optical Configuration Typical Fluorochromes 405 530/40 400 450 500 488 nm 488 550 450 500 600 650 530/40 575/25 613/20 FL1 400 DAPI Alexa 405 Pacific Blue Violet 2 (FL7) Violet 1 (FL6) 405 nm 450/50 550 FL2 700 680/30 FL3 600 750 700 FL5 750 642 500 550 600 650 PE PI PI PE-Texas Red PE-Alexa 610 PI PE-Cy5 PerCP PerCP-Cy5.5 800 APC-Cy7 (FL9) 642 nm 450 GFP FITC Alexa488 CFSE 750LP APC (FL8) 665/20 400 800 750LP FL4 650 Alexa 430 AmCyan Pacific Orange 700 750 800 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* PE-Cy7 Know Your Instrument (FACSAria) FACSAria Optical Configuration Typical Fluorochromes 407 530/30 530/30 400 450 500 600 650 585/42 616/23 PE FITC 488 nm 488 550 550 700 600 750 695/40 650 700 780/60 750 633 450 500 550 600 650 GFP FITC Alexa488 CFSE PE PI PI PE-Texas Red PE-Alexa 610 PI PE-Cy5 PerCP PerCP-Cy5.5 800 APC-Cy7 780/60 APC 633 nm 660/20 400 Alexa 430 AmCyan Pacific Orange 800 PE-Cy7 500 PerCP-Cy5.5 450 PE-Texas Red 400 DAPI Alexa 405 Pacific Blue Alexa 430 DAPI 407 nm 450/40 700 750 800 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* PE-Cy7 Know Your Instrument (MoFlo) MoFlo Optical Configuration #5 PE-Cy75 #6 795/50 PE-TxRed APC 616/26 #4 SSC Red #8 D405/30 670/30 616/26 #1 FITC H-Blue 530/40 #2 PE 488/10 #3 #7 585/40 670/40 Blue Yellow UV mCherry H-Red #9 Know Your Instrument (LSR Fortessa) LSR Fortessa Optical Configuration 488 nm (Blue) 561 nm (YG) 442 nm (BV) Know Your Instrument (LSR Fortessa) PE-Cy5, PE-A647 mPlum PE RFP, DsRed dTomato mOrange YG:561nm YELLOW GREEN (561 nm) PE-Cy7 630/75 or 590LP in position A For mCherry detection only PE-TexasRed PI mCherry mRaspberry mplum Know Your Instrument (LSR Fortessa) BLUE VIOLET (442 nm) 455LP 442 nm (BV) Know Your Instrument (LSR Fortessa) PE-Cy5 Blue:488nm SSC BLUE (488 nm) PE-Cy7 FITC Alexa 488 GFP YFP Know Your Instrument (LSR Fortessa) YFP Blue:488nm SSC BLUE (488 nm) PE-Cy7 GFP Measure GFP and YFP simultaneously Choosing The Right Fluorochromes Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34 Rule 1 Choose the Brightest Fluorochromes Fluorochromes (Stain Index) Brightest Fluorochrome = Highest Stain Index D Stain Index = W1 W2 Stain Index (SI) =D/W MFIPOSITIVE ̶ MFINEGATIVE 2 × rSDNEGATIVE Fluorochromes (Stain Index) Freshly isolated lymphocytes, stained with anti-human CD3 antibodies conjugated with various fluorochromes Fluorochromes (Choose the brightest) Stain Index of various anti-CD4 fluorochrome conjugates measured on a BD LSR II Reagent Clone Filter Stain Index PE RPA-T4 585/40 356.3 Alexa 647 RPA-T4 660/20 313.1 APC RPA-T4 660/20 279.2 PE-Cy7 RPA-T4 780/60 278.5 PE-Cy5 RPA-T4 695/40 222.1 PerCP-Cy5.5 Leu-3a 695/40 92.7 PE-Alexa 610 RPA-T4 610/20 80.4 Alexa 488 RPA-T4 530/30 75.4 FITC RPA-T4 530/30 68.9 PerCP Leu-3a 695/40 64.4 APC-Cy7 RPA-T4 7801/60 42.2 Alexa 700 RPA-T4 720/45 39.9 Pacific Blue RPA-T4 440/40 22.5 AmCyan RPA-T4 525/50 20.2 Rule 2 Minimize Potential Spillover Spillover (Minimize spillover) A single fluorochrome can be detected in more than one channel Spectral Overlap Correcting spillover Compensation Compensation is a mathematical subtraction to correct spectral overlap A488true = A488measured - % 1PE true 0%%A488 5 10 15 20 % true PE true = PE measured - 30 1000 Alexa488 100 10 1 1 10 100 PE http://www.drmr.com/compensation/ Roederer, M. 2002. Compensation in Flow Cytometry. Current Protocols in Cytometry. 1.14.1–1.14.20 1000 Spillover (Minimize spillover) Slide taken from presentation: Mario Roederer, “Compensation: Basic Principles”, Monday, June 20, 2011 Rule 3 Reserve brightest fluorochromes for “dim” antibodies and vice-versa Colors and Antibody Specificities (Reserve bright labels for dim antibodies) Single Stain Controls CD4-PE Fluorescence CD4-Alexa488 Fluorescence Single Stain Controls CD25-PE Fluorescence CD4-PE Fluorescence CD4-Alexa488 Fluorescence CD25-Alexa488 Fluorescence CD25-Alexa488 Fluorescence CD25-PE Fluorescence Rule 4 Avoid spillover from bright populations into detectors requiring high sensitivity Colors and Antibody Specificities (Avoid spillover of bright cells into detectors of dim signals) Sample CD4-Alexa488 Fluorescence CD4-Cy5 Fluorescence CD4-Alexa488 CD4-Cy5 Fluorescence Fluorescence Single Stain Controls CD4-Alexa488 Fluorescence CD25-PE Fluorescence CD25-PE Fluorescence CD25-PE Fluorescence Rule 5 Take steps to avoid tandem dye degradation Tandem Dyes Watch out for degradation TIME APC Fluorescence APC-50% PE-Cy5 PE-Cy5 Fluorescence APC- 40% PE-Cy5 PE-Cy5 Fluorescence PE-Cy5 Fluorescence APC- 30% PE-Cy5 APC Fluorescence PE-Cy5 PE-Cy7 APC-Cy7 APC Fluorescence APC-H7 “Rules” for selecting Multicolor Panel taken from BD Biosciences Rule 1: Choose the brightest set of fluorochromes for your particular instrument configuration. Rule 2: Choose fluorochromes so as to minimize the potential for spillover. Rule 3: Reserve the brightest fluorochromes for “dim” antibodies, and vice versa. Rule 4: Avoid spillover from bright cell populations into detectors requiring high sensitivity for those populations. Rule 5: Take steps to avoid tandem dye degradation, and consider its impact upon results. Recommended Multicor Panel Fluorochrome choices for 5 or more colors (Recommended by BD) 5-color 6-color 8-color 10-color FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 PE PE PE PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5 PerCP-Cy5.5 PE-Cy7 PE-Cy7 PE-Cy7 PE-Cy7 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 Alexa 700 APC-Cy7 APC-Cy7 APC-Cy7 AmCyan AmCyan Pacific Blue Pacific Blue Recommended Multicor Panel Fluorochrome choices for 5 or more colors (Recommended by IGC) 5-color 6-color 8-color 10-color FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 FITC or Alexa 488 PE PE PE PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 or PerCP APC , Alexa 647 or Cy5 PerCP-Cy5.5 or PerCP PerCP-Cy5.5 or PerCP PerCP-Cy5.5 or PerCP PE-Cy7 PE-Cy7 PE-Cy7 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 APC , Alexa 647 or Cy5 Alexa 700 Pacific Blue Pacific Blue APC-Cy7 APC-Cy7 Pacific Orange Pacific Orange Pacific Blue Pacific Blue What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry (end) IGC – Aprli 03, 2013