Presentation is loading. Please wait.

Presentation is loading. Please wait.

What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 03, 2013 Adapted from Holden.

Similar presentations


Presentation on theme: "What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 03, 2013 Adapted from Holden."— Presentation transcript:

1 What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 03, 2013 Adapted from Holden and Trotter (Winter 2006) Selecting Reagents for Multicolor Flow Cytometry BD Hotlines newsletter, 11:

2 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

3 Know Your Instrument

4 Reagent Selection starts with Instrument Configuration Lasers Detectors and respective filters Analyzers Cell Sorters FACSCalibur FACScan MoFlo FACSAria CyAn ADP HyperCyt LSR Fortessa

5 Know Your Instrument (FACScan) FACScan Optical Configuration /30585/42 650LP FL1FL2FL3 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 Typical Fluorochromes 488 nm

6 Know Your Instrument (FACSCalibur) FACSCalibur Optical Configuration Typical Fluorochromes /30585/42 670LP FL1FL2FL3 488 nm 633 nm /16 FL4 APC Cy5 Alexa647 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610

7 Know Your Instrument (CyAn ADP) CyAn ADP Optical Configuration GFP FITC Alexa488 CFSE PE PI PI PE-Cy5 PerCP PerCP-Cy5.5 Typical Fluorochromes 405 nm 642 nm APC Cy5 Alexa nm / Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 Pacific Blue PI PE-Texas Red PE-Alexa 610 PE-Cy7 APC-Cy7 APC-H7 Alexa 700* 530/40 575/25613/20680/30750LP 665/20 FL1FL2FL3FL4FL5 APC (FL8) APC-Cy7 (FL9) Violet 1 (FL6) Violet 2 (FL7)

8 Know Your Instrument (FACSAria) FACSAria Optical Configuration GFP FITC Alexa488 CFSE PE PI PI PE-Cy5 PerCP PerCP-Cy5.5 Typical Fluorochromes 407 nm 633 nm APC Cy5 Alexa nm /40 DAPI Alexa FITC PE PE-Texas Red PerCP-Cy5.5 PE-Cy APC APC-Cy Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 Pacific Blue PI PE-Texas Red PE-Alexa 610 PE-Cy7 APC-Cy7 APC-H7 Alexa 700* 530/30 585/42616/23695/40780/60 660/20

9 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 Know Your Instrument (MoFlo) MoFlo Optical Configuration H-Blue H-Red UV 670/ DCLP D405/30 Yellow mCherry 616/26

10 Know Your Instrument (LSR Fortessa) LSR Fortessa Optical Configuration 442 nm (BV) 561 nm (YG) 488 nm (Blue)

11 Know Your Instrument (LSR Fortessa) YELLOW GREEN (561 nm) YG:561nm PE-TexasRed PI mCherry mRaspberry mplum 630/30 780/60 670/40 590/30 610LP 740LP 650LP PE-Cy7 630/75 or 590LP in position A For mCherry detection only PE-Cy5, PE-A647 mPlum PE RFP, DsRed dTomato mOrange

12 Know Your Instrument (LSR Fortessa) 445/15 470/20 577LP 455LP SSC (BV) CFP 442 nm (BV) BLUE VIOLET (442 nm)

13 Know Your Instrument (LSR Fortessa) BLUE (488 nm) Blue:488nm FITC Alexa 488 GFP YFP 530/30 780/60 690/40 488/10 502LP 740LP 655LP PE-Cy7 PE-Cy5 SSC

14 Know Your Instrument (LSR Fortessa) Blue:488nm GFP 510/20 780/60 540/30 488/10 502LP 740LP 525LP PE-Cy7 YFP SSC BLUE (488 nm) Measure GFP and YFP simultaneously

15 Choosing The Right Fluorochromes Adapted from Holden and Trotter (Winter 2006) Selecting Reagents for Multicolor Flow Cytometry BD Hotlines newsletter, 11:

16 Rule 1 Choose the Brightest Fluorochromes

17 Fluorochromes (Stain Index) Brightest Fluorochrome = Highest Stain Index W2 W1 D Stain Index (SI) =D/W MFI POSITIVE MFI NEGATIVE ̶ 2 × rSD NEGATIVE Stain Index =

18 Fluorochromes (Stain Index) Freshly isolated lymphocytes, stained with anti-human CD3 antibodies conjugated with various fluorochromes

19 Fluorochromes (Choose the brightest) Stain Index of various anti-CD4 fluorochrome conjugates measured on a BD LSR II

20 Rule 2 Minimize Potential Spillover

21 Spillover (Minimize spillover) A single fluorochrome can be detected in more than one channel Spectral OverlapCorrecting spillover

22 A488 true = A488 measured - % PE true PE true = PE measured - % A488 true 0 %5 %10 %15 %20 %30 % Compensation Compensation is a mathematical subtraction to correct spectral overlap Alexa488 PE A488 true = A488 measured - % 1 Roederer, M Compensation in Flow Cytometry. Current Protocols in Cytometry –

23 Spillover (Minimize spillover) Slide taken from presentation: Mario Roederer, Compensation: Basic Principles, Monday, June 20, 2011

24 Rule 3 Reserve brightest fluorochromes for dim antibodies and vice-versa

25 Colors and Antibody Specificities (Reserve bright labels for dim antibodies) Single Stain Controls CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD4-PE Fluorescence CD25-Alexa488 Fluorescence Single Stain Controls CD25-Alexa488 Fluorescence

26 Rule 4 Avoid spillover from bright populations into detectors requiring high sensitivity

27 Sample CD25-PE Fluorescence CD4-Alexa488 Fluorescence Single Stain Controls CD4-Alexa488 Fluorescence CD25-PE Fluorescence Colors and Antibody Specificities (Avoid spillover of bright cells into detectors of dim signals) CD4-Cy5 Fluorescence CD25-PE Fluorescence

28 Rule 5 Take steps to avoid tandem dye degradation

29 Tandem Dyes Watch out for degradation APC Fluorescence PE-Cy5 Fluorescence TIME PE-Cy5 PE-Cy7APC-Cy7APC-H7 APC- 30% PE-Cy5APC- 40% PE-Cy5APC-50% PE-Cy5

30 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.

31 Recommended Multicor Panel 5-color6-color8-color10-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 Fluorochrome choices for 5 or more colors (Recommended by BD)

32 Recommended Multicor Panel 5-color6-color8-color10-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 Fluorochrome choices for 5 or more colors (Recommended by IGC)

33 What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry (end) IGC – Aprli 03, 2013


Download ppt "What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 03, 2013 Adapted from Holden."

Similar presentations


Ads by Google