بسم الله الرحمن الرحیم

Flow Cytometry

فلو سایتو متری پروسه ای است که طی آن سلولها یا دیگر ذرات بیولوژیک بصورت انفرادی از داخل یک جریان سیال عبور داده می شوند و مجموعه ای از گیرنده های حساس ،خصوصیات فیزیکی یا شیمیائی آنها را اندازه گیری می کند

Publications using the keyword “flow cytometry” from 114908 references (2010) 1st The growth of diagnostic and phenotypic determination technologies

Papers Published per year in: 1400 Papers Published per year in: 1200 1000 800 Papers 600 400 200 1970 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 Year

Flow Cytometry FACS – Fluorescence Activated Cell Sorter is the generic term used for flow cytometry (even without sorting) Simultaneous analysis of different physical parameters in a single cell Can analyze up to several thousands of cells per second Versatile, sensitive

What is in a Flow Cytometer? Fluidics To introduce and focus the cells for interrogation by a laser Optics To generate and collect the light signals (scatter and fluorescence) Electronics To convert the optical signals to proportional electronic signals and digitize them for computer analysis (PMTs)

آنالیز نمونه مراحل 1-آماده سازی سوسپانسیون سلولی 2-نشانه گذاری سلولها به وسیله پروپ (آنتی بادی مونوکلونال نشانه گذاری شده) 3-آنا لیز دستگاه 4- جمع آوری ،ذخیره سازی وآنالیز اطلاعات 5-تفسیر وگزارش اطلاعات بدست آمده

Incident Light Source FSC Detector SSC Detector

Light can be measured at 90° : Side scatter + Fluorescence Photodiode Laser Side scatter reflects the cell content

CD8-FITC CD3-PerCP CD62L-APC Perforin-PE

Flow Cytometry Optics Dichroic Filters Flow cell Bandpass Filters PMT 4 Dichroic PMT Filters 3 Flow cell PMT 2 Bandpass Filters PMT 1 Laser

Standard Band Pass Filters 630 nm BandPass Filter White Light Source Transmitted Light 620 -640 nm Light

Optical Filters Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light

Standard Long Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light Standard Short Pass Filters 575 nm Short Pass Filter Light Source Transmitted Light <575 nm Light

Photomultiplier tubes (PMT’s) The PMTs in an Elite. 3 PMTs are shown, the other 2 have been removed to show their positions. A diode detector is used for forward scatter and a PMT for side scatter. The Bio-Rad Bryte cytometer uses PMTs for forward and wide angle light scatter as well as fluorescence

Signal Detection - PMTs Secondary emission Cathode Anode Photons in Amplified Signal Out End Window Dynodes

Types of PMTs Side Window Signal out High voltage in

Whole Blood-RBCs lysed Largest and most complex population 1000 Side Scatter Forward Light Scatter 200 400 600 800 Smallest and least complex population Neutrophils Eosinophils Monocytes Lymphocytes

Fluorochromes: Excitation and Emission Spectra Antibodies can be conjugated to fluorochromes The amount of fluorescent signal detected is proportional to the number of fluorochrome molecules on the particle.

Fluorescence intensity Relative fluorescence intensity FITC FITC FITC FITC FITC FITC FITC FITC Number of Events FITC FITC 101 102 103 104 Relative fluorescence intensity 6

Fluorescence Channels Fluorochromes FL1 FITC, Alexa 488, GFP, CFSE FL2 PE, DsRed, Alexa 594, PI (DNA) FL3 PE-Cy5, PE-Cy7 tandem conjugates, PerCP, PerCP-Cy5 PI, 7-AAD (DNA) FL4 APC, Cy5, DDAO-SE, ToPro-3 (DNA)

Flow Cytometry Analysis Single parameter analysis: Histogram plot Horizontal axis: level of fluorescence - brighter cells further right Vertical axis: number of events per channel number Analyze level of expression of marker Two parameter analysis: Dot-plot One axis shows first color Second axis shows second color Analysis of individual populations of cells 10 1 2 3 4 CD86 PE Isotype MUTZ-3 iDC mDC.031

COLOR PATTERNS USED TO VIEW COEXPRESSION b 2 1 3 c o l r ne eg . ( - ) n e g y p s + u t w a v i no d When three antibodies are used together the eight possible combinations of positive and negative cells are shown in the table. When a unique color is assigned to each combination, the position of cells with the same properties generate patterns in the three bivariate plots, representing all the combinations.

Types of Antigen Expression B C Ab2 Ab2 Ab2 Ab1 Ab1 Ab1 Ab1 D E F There are several patterns of antigen expression. The interpretation of them is important for describing the meaning of co-expression. In A, Ab2 is uniformly expressed, while Ab1 is co-expressed by Ab2 cells in a heterogeneous manner. In B, Ab1 is uniformly expressed, while Ab2 is co-expressed by Ab1 cells in a heterogeneous manner. In C, a population of Ab1+Ab2- cells is found, but it is the Ab2+ cells that exhibit heterogeneous expression to produce the Ab1+Ab2+ population. Thus, Ab1 cells are not positive for Ab2. In D, there are two distinct populations: Ab2+Ab1- and Ab2+Ab1+, while in E, the two populations are Ab1+Ab2- and Ab1+Ab2+. In F, correct interpretation can be difficult because this pattern is exhibited by highly autofluorescent cells that are Ab1-Ab2- or dead cells that non specifically bind Ab1 and Ab2. If it is either of these two situations, all combinations of antibodies used will show this pattern on the same percentage of cells. If this pattern represents a true subpopulation of cells it will be unique to the one antibody combination. Ab2 Ab2 Ab2 Ab1 Ab1 Ab1

Common Apllications of Flow Cytometry in Immunology Phenotype of cell, surface molecules Intracellular cytokine staining Antigen specificity Cell proliferation (e.g. CFSE, BrdU incorporation) Cell sorting Apoptosis analysis Cytotoxicity assays Phagocytosis assays Cell cycle analysis (DNA content analysis) Cell signalling molecules, Calcium flux assays Organelle-specific studies (e.g. lysosome) Cellular transport assays Transfection efficiencies

Intracellular Cytokine Staining To detect cytokine production by a specific cell upon stimulation Used to define T cell activation by epitope recognition and the T cell polarization

Normal Cell Cycle s s M G2 G2 M G0 G1 G0 G1 2N 4N DNA content DNA Analysis G1 G0 G1 s Count s G2 M 2N 4N 200 400 600 800 1000 DNA content

DNA Analysis / Apoptosis 12 Annexin V PI Ploidy determination, detection of abnormal clones. Cell cycle analysis. Apoptosis. Flow karyotyping (chromosome analysis).

Cell Sorting - FACS

آنالیزاطلاعات غیر پارامتریک پارامتریک احتمال توضیح متفاوت دو فلورسانس مختلف کانال به کانالt مانند تست وآنالیز kolmogorov-Smirnov پارامتریک *میانگین فلورسانس شماره کانال *میانگین فلورسانس سلولهای مثبت CV *ضریب تغییرات

Sources of information Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, M.L. Mendelsohn, eds.), Wiley-Liss, New York, 1990 - referred to here as MLM Flow Cytometry: Instrumentation and Data Analysis (M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R. Melamed, eds.), Academic Press, London, 1985 – referred to as VDLM Practical Flow Cytometry 3nd edition (1994),H. Shapiro: Alan R. Liss, New York - referred to as PFC Introduction to Flow Cytometry. J. Watson, Cambridge Press, 1991 referred to as IFC Methods in Cell Biology: v.40,41, 63, 64 Darzynkiewicz, Robinson & Crissman, Academic Press, 1994, 2000 MCB Data Analysis in Flow Cytometry:A Dynamic Approach-Book on CDROM M. Ormerod referred to as DAFC Flow Cytometry: First Principles. (2nd Ed) Alice Longobardi Givan, Wiley-Liss, 2001 referred to as AFCFP Sources This slide is intended to show students supplementary materials if they are interested in learning more about flow cytometry. More information on flow cytometry books can be found on our website at: http://www.cyto.purdue.edu/flowcyt/books/bookindx.htm Note: All of these books are in Prof. Robinson’s library in Hansen Hall, Room B50 and may be checked out for 24 hour periods with permission. ©J.Paul Robinson, Purdue University BMS 631 - LECTURE1.PPT