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Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry Rui Gardner IGC – April 03, 2013 Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11:
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Outline Know Your Instrument Choosing the right fluorochromes
Optical Layout (lasers and filters) Choosing the right fluorochromes Staining Index Spillover Compensation Color Specificities and Tandem Dyes Rules for Multicolor Analysis
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Know Your Instrument
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Know Your Instrument Reagent Selection starts with Instrument Configuration Lasers Detectors and respective filters FACScan FACSCalibur CyAn ADP Analyzers HyperCyt FACSAria MoFlo Cell Sorters LSR Fortessa
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Know Your Instrument (FACScan)
FACScan Optical Configuration Typical Fluorochromes 488 400 450 500 550 600 650 700 750 800 530/30 585/42 650LP FL1 FL2 FL3 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 488 nm
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Know Your Instrument (FACSCalibur)
FACSCalibur Optical Configuration Typical Fluorochromes 488 530/30 585/42 670LP 400 450 500 550 600 650 700 750 800 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 FL1 FL2 FL3 488 nm 633 400 450 500 550 600 650 700 750 800 661/16 FL4 APC Cy5 Alexa647 633 nm
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Know Your Instrument (CyAn ADP)
CyAn ADP Optical Configuration Typical Fluorochromes 405 450/50 530/40 400 450 500 550 600 650 700 750 800 DAPI Alexa 405 Pacific Blue Alexa 430 AmCyan Pacific Orange 405 nm Violet 1 (FL6) Violet 2 (FL7) 488 530/40 575/25 613/20 680/30 750LP 400 450 500 550 600 650 700 750 800 GFP FITC Alexa488 CFSE PE PI PI PE-Texas Red PE-Alexa 610 PI PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7 488 nm FL1 FL2 FL3 FL4 FL5 642 665/20 750LP 400 450 500 550 600 650 700 750 800 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* APC (FL8) APC-Cy7 (FL9) 642 nm
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Know Your Instrument (FACSAria)
FACSAria Optical Configuration Typical Fluorochromes 407 450/40 530/30 400 450 500 550 600 650 700 750 800 DAPI Alexa 405 Pacific Blue Alexa 430 AmCyan Pacific Orange 407 nm DAPI Alexa 430 488 530/30 585/42 616/23 695/40 780/60 400 450 500 550 600 650 700 750 800 GFP FITC Alexa488 CFSE PE PI PI PE-Texas Red PE-Alexa 610 PI PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7 488 nm FITC PE PE-Texas Red PerCP-Cy5.5 PE-Cy7 633 660/20 780/60 400 450 500 550 600 650 700 750 800 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* 633 nm APC APC-Cy7
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Know Your Instrument (MoFlo)
MoFlo Optical Configuration SSC FITC PE PE-Cy75 #1 #2 #3 #4 #5 #6 #7 #8 #9 Blue Red 488/10 530/40 585/40 616/26 95/5BS 555DLP 610DLP 645DLP PE-TxRed 795/50 670/40 APC Yellow mCherry 616/26 H-Blue H-Red UV 670/30 565 DCLP D405/30
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Know Your Instrument (LSR Fortessa)
LSR Fortessa Optical Configuration 488 nm (Blue) 561 nm (YG) 442 nm (BV)
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Know Your Instrument (LSR Fortessa)
PE-Cy5, PE-A647 mPlum PE RFP, DsRed dTomato mOrange YG:561nm YELLOW GREEN (561 nm) 670/40 590/30 650LP 610LP 740LP 780/60 630/30 PE-Cy7 PE-TexasRed PI mCherry mRaspberry mplum 630/75 or 590LP in position A For mCherry detection only
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Know Your Instrument (LSR Fortessa)
BLUE VIOLET (442 nm) SSC (BV) 445/15 577LP 455LP 470/20 CFP 442 nm (BV)
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Know Your Instrument (LSR Fortessa)
PE-Cy5 Blue:488nm SSC BLUE (488 nm) 690/40 488/10 655LP 502LP 740LP 780/60 530/30 PE-Cy7 FITC Alexa 488 GFP YFP
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Know Your Instrument (LSR Fortessa)
YFP Blue:488nm SSC BLUE (488 nm) 540/30 488/10 525LP 502LP 740LP 780/60 510/20 PE-Cy7 GFP Measure GFP and YFP simultaneously
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Choosing The Right Fluorochromes
Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11:
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Choose the Brightest Fluorochromes
Rule 1 Choose the Brightest Fluorochromes
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Fluorochromes (Stain Index)
Brightest Fluorochrome = Highest Stain Index D W2 W1 MFIPOSITIVE MFINEGATIVE ̶ 2 × rSDNEGATIVE Stain Index = Stain Index (SI) =D/W
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Fluorochromes (Stain Index)
Freshly isolated lymphocytes, stained with anti-human CD3 antibodies conjugated with various fluorochromes
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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 647 660/20 313.1 APC 279.2 PE-Cy7 780/60 278.5 PE-Cy5 695/40 222.1 PerCP-Cy5.5 Leu-3a 92.7 PE-Alexa 610 610/20 80.4 Alexa 488 530/30 75.4 FITC 68.9 PerCP 64.4 APC-Cy7 7801/60 42.2 Alexa 700 720/45 39.9 Pacific Blue 440/40 22.5 AmCyan 525/50 20.2 However stain indexes are calculated with reagents run on their own, and not part of a cocktail
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Minimize Potential Spillover
Rule 2 Minimize Potential Spillover
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Spillover (Minimize spillover)
A single fluorochrome can be detected in more than one channel Spectral Overlap Correcting spillover
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Compensation Compensation is a mathematical subtraction to correct spectral overlap A488true = A488measured - % PE true PE true = PE measured - % A488true A488true = A488measured - % 1 20 % 30 % 15 % 0 % 5 % 10 % Alexa488 PE Roederer, M Compensation in Flow Cytometry. Current Protocols in Cytometry –
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Spillover (Minimize spillover)
Slide taken from presentation: Mario Roederer, “Compensation: Basic Principles”, Monday, June 20, 2011
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Reserve brightest fluorochromes for “dim” antibodies and vice-versa
Rule 3 Reserve brightest fluorochromes for “dim” antibodies and vice-versa
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Colors and Antibody Specificities
(Reserve bright labels for dim antibodies) CD4-PE Fluorescence CD25-Alexa488 Fluorescence Single Stain Controls Single Stain Controls CD25-PE Fluorescence CD4-Alexa488 Fluorescence
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Rule 4 Avoid spillover from bright populations into detectors requiring high sensitivity
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Colors and Antibody Specificities
(Avoid spillover of bright cells into detectors of dim signals) Single Stain Controls Sample CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD4-Cy5 Fluorescence CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD25-PE Fluorescence {{ ,2000},{ ,2000}}, PE vs A488 with PEtrue=PEmeas-10%A488True CD4-Alexa488 Fluorescence CD25-PE Fluorescence
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Take steps to avoid tandem dye degradation
Rule 5 Take steps to avoid tandem dye degradation
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Tandem Dyes Watch out for degradation TIME PE-Cy5 PE-Cy7 APC-Cy7
APC- 30% PE-Cy5 APC- 40% PE-Cy5 APC-50% PE-Cy5 PE-Cy5 Fluorescence PE-Cy5 Fluorescence PE-Cy5 Fluorescence APC Fluorescence APC Fluorescence APC Fluorescence PE-Cy5 PE-Cy7 APC-Cy7 APC-H7
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“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.
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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 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 PE-Cy7 APC , Alexa 647 or Cy5 Alexa 700 APC-Cy7 AmCyan Pacific Blue
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Completely Outdated! 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 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 or PerCP PE-Cy7 APC , Alexa 647 or Cy5 Alexa 700 APC-Cy7 Pacific Orange Pacific Blue Completely Outdated!
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Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry (end) Rui Gardner IGC – Aprli 03, 2013
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