Introduction to the Principles of Flow Cytometry for users of the Flow Cytometry Core Facility at BUMC 21 September 2010 Mike Xie (X4-5225), mike.xie@bmc.org Yan Deng (X4-5225), ydeng@bu.edu John Meyers (X8-7543), johnm@bu.edu Gerald Denis (X4-1371), gdenis@bu.edu

Flow Cytometry Core Facility: Personnel Director of Cell Sorting Services: Yanhui Deng Operations Manager: Mike Xie Facility Consultant: John Meyers Co-Director/Outreach: Gerald Denis Director: David Sherr http://www.bu.edu/cores/flow-cytometry

Where are the instruments located? LSR II, MoFlo:670-5 FACScan: R901 X620 FACScan, FACSCalibur:L508 U R here http://www.bu.edu/cores/flow-cytometry

Flow cytometry was not easy in the old days ungloved hands missing PPE gendered division of labor live anthrax bacilli

But today… it’s as easy as PCR.

3 elements of any flow cytometry system fluidics optics electronics

fluidics

Fluidics sample fluid Purpose of the fluidics system: sheath fluid Purpose of the fluidics system: Transport particles in a fluid stream to the laser beam to be interrogated 2. Position the sample core in the center of the laser beam ‘hydrodynamic focusing’ single file particles ● low flow rate ● narrow sample core ● high resolution ● high flow rate ● wide sample core ● low resolution Always filter your samples to remove aggregates.

Fluidics When conditions are right (i.e. when turbulence is minimal): sample fluid flows in a central core does not mix with the sheath fluid This is termed ‘laminar flow’

optics electronics “SSC” “FSC”

Forward and side scatter of leukemic cells Normal B cells Malignant, large B cells SSC FSC The intensity of forward scatter light is proportional to size and cross-sectional area of the cells. The intensity of side scatter light is proportional to size, shape and internal structure/irregularity of the cells.

Photomultiplier (PMT) detectors convert photons (selected by mirrors and filters) to electrical pulses Peak Height (volts) Peak Width (time) Peak Area The higher the PMT voltage (user controllable), the greater the output magnitude for a given photon. At higher PMT voltages, the level of noise will also increase.

Photomultiplier Tubes (PMTs) FSC: forward scatter (size; cross-sectional area) SSC: side scatter (granularity, internal or surface structure that scatters light) Adjusting the voltage of the PMT helps to optimize capture of desired populations

A dotplot represents two properties of a single cell SSC FSC

Control Condition Experimental A histogram represents the distribution of a single parameter across many cells 10,000 cells each! Control Condition Experimental 1 cell 1 cell 1 cell 1 cell

Electronic processing of emission signals Amplifiers are of two types: linear or logarithmic Linear amplification is typically used with scatter. Logarithmic amplification is typically used with fluorescence. DNA content (Linear detection) (Log detection)

Gating Gating allows one to select populations based on computer or human-derived criteria and further gate or display the included cells

Backgating – don’t lose your bearings! Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type. In the above example, some cells are missed! What are they? Many investigators overlook the importance of verification by backgating!

FLUORESCENCE Excitation wavelength and emission wavelength are blue laser Excitation wavelength and emission wavelength are unique properties of each specific molecular structure (FITC)

Stokes Fluorescence Excitation Emission Stokes shift

yes no Laser light must overlap with excitation wavelength Fluorescein (FITC) Propidium iodide (PI) 488 488 ex em yes ex em Hoechst 33258 Texas Red 488 488 no ex em ex em

Widely-used molecules are excited by the 488 nm laser (FACScan) 488 nm (Blue) : FITC, GFP, PE, PerCP, PE-Cy5, PI, PerCP-Cy5.5, PE-Cy7 But different lasers are available to excite other molecules (LSR II) 355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258 405 nm (Violet) : Alexa Fluor 430, Alexa Fluor 405, Pacific Blue 561 nm (Yellow/Green): Texas Red, Cherry Red, Tomato Red 633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680

Octagon Detector Arrays emitted fluorescent light longpass dichroic mirrors bandpass filters

EMISSION

Fluorescence detection autofluorescence weakly expressed epitope strongly expressed epitope Note logarithmic scale

Isotype controls weakly expressed epitope isotype control test How do you know it’s real? weakly expressed epitope isotype control test Isotype control antibodies should be used at the same concentration to stain cells at the same cell density as the experimental, but they give fluorescent signals that define a negative result.

Resolution sensitivity Resolution sensitivity, the ability to resolve a faint signal from background) depends on the difference D between the positive and background peaks and the spread of the background peak W

Choose the right fluor for the job! Reagent Stain Index Phycoerythrin (PE) 356.3 Alexa Fluor 647 313.1 APC 279.2 PE-Cy7 278.5 PE-Cy5 222.1 PerCP-Cy5.5 92.7 PE-Alexa Fluor 610 80.4 Alexa Fluor 488 75.4 FITC 68.9 PerCP 64.4 APC-Cy7 42.2 Alexa Fluor 700 39.9 Pacific Blue 22.5 AmCyan 20.2 i.e. pick a bright fluor for a dim epitope and avoid spillover of bright cell populations into detector channels that require high sensitivity for rare signals

Problems in Emission Fluorescence Excitation Emission Spectral overlap

Optical solutions to spectral overlap: Filters Filters resolve overlapping wavelengths of emitted light Longpass filter: transmits light of longer than or equal to a specific wavelength Shortpass filter: transmits light of shorter than or equal to Bandpass filter: transmits light only within a narrow range of wavelengths

Examples of optical filters in flow cytometry

Optical detector configurations octagon red trigon 660/20 APC 735 LP 780/60 APC-Cy7 bandpass longpass bandpass

EMISSION two bandpass filters

Electronic solutions to spectral overlap: Compensation To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both. Uncompensated Optimal

COMPENSATION Multicolor immunophenotyping Before After

No antibody. Autofluorescence only. No compensation applied.

CD4-PE. No compensation applied.

CD4-PE. Correct compensation applied. 1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.

CD8-FITC. No compensation applied.

CD8-FITC. Correct compensation applied. 12.5% FITC subtracted from PE PMT.

CD4-PE + CD8-FITC. Streptavidin-APC alone.

CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC 3 colors, correctly compensated

Therefore, avoid such combinations Spectral overlap of some fluorochrome combinations cannot be compensated easily or at all APC Cy5 Therefore, avoid such combinations

Contour plots provide more accurate data representation than dot plots

Gates granularity → size →

The uses of gates for cell sorting SSC FSC CD8 CD3

Four Applications Cell cycle analysis Multicolor immunophenotyping Phosphoprotein and kinase signaling Stem cell sorting by the “side population” method

DNA content (propidium iodide) Cell cycle analysis G0/G1 G2/M S S anti-BrdU-FITC (DNA synthesis) G2/M G0/G1 2N 4N 2N 4N DNA content (propidium iodide) 7-aminoactinomycin D Linear detection

Multicolor, Auto Compensation with Flow Jo 20 40 60 80 100 % of Max isotype 20 40 60 80 100 % of Max isotype gate 1: lymphocytes gate 2: B cells 10 1 2 3 4 10 1 2 3 4 gate 6: T cells? FITC Pacific Blue 20 40 60 80 100 % of Max isotype 20 40 60 80 100 % of Max isotype etc 200 400 600 800 1000 10 1 2 3 4 gates 3 – 5 10 1 2 3 4 10 1 2 3 4 PE PE-Cy7 up to 11 colors

Filter configurations permit optimization of multicolor stains

When two colors are not enough A single PBMC sample simultaneously stained with antibodies to quantify expression of CD3, CD4, CD8, CD7, CD27, CD28, CD45RA, CD62L and CCR7. A lymphocyte size gate and CD3+/CD4-/CD8+ color gate is applied to characterize stages of T cell differentiation. The T cell compartment cannot be fully characterized by only 2 or 3 markers. Current Protocols in Immunology 12:12 (2005)

Example of a 10-color experiment on our LSR II Other markers Fluorochrome Marker B220: B lineage IgM: mature B cells IgD: mature B cells CD23: transitional B cells AA4.1: transitional B cells Pacific Blue PE-Cy7 PE FITC APC CD45.1: donor phenotype CD11b/Mac-1: myeloid lineage live/dead discrimination* CD45.2-Biotin: recipient phenotype CD3: T lineage PerCP-Cy5.5 APC-Cy7 Invitrogen UV-activated vital dye Streptavidin Alexa 350 Qdot 605

FITC Compensation Matrix FITC FITC

“Phosphoflow” techniques allow you to measure kinase cascades and signal transduction Courtesy John Meyers johnm@bu.edu

Identification of a Hoechst 33342-staining ‘side population’ from murine bone marrow Hoechst red Hoechst blue 0.25% Control Verapamil 100% of the gated side population is also Sca1+ ; these are hematopoietic stem cells.

Kinetic identification of “side population” stem cells These gated stem cells can be isolated by MoFlo

FCCF rates http://www.bu.edu/cores/flow-cytometry On-line scheduling Actual cytometer and work station use tracked to the minute, recorded in and billed monthly by enterprise class central server http://www.bu.edu/cores/flow-cytometry

Problems during experiments: Help and training: Please sign up for basic training on FACScan or LSR2 with Yan Deng (X4-5225), ydeng@bu.edu Problems during experiments: We can’t read minds. Please write a computer entry in the COMPLAINT LOG for each instrument. Or email us: flowcore@bu.edu We will contact you ASAP and usually can respond within 2 hours.