FLOW CYTOMETRY
PRESENTED BY
PRATIK PUROHIT
M PHARM PHARMACOLOGY
2025063105
CONTEXT
INTRODUCTION
PRINCIPLE
WORKING
APPLICATION
INTRODUCTION
Laser based technique
Detect & measure physical and chemical feature
of cell .
In a heterogenous fluid mixture or suspending
Help to identify-
media. [Link] size
[Link]
[Link] Intensity
PRINCIPLE
HYDRODYNAMIC
FOCUS
•Helps to align the cell
or
•Maintain laminar flow of
cell suspension.
•Done by sheath fluid which
is faster moving liquid with
uniform pressure,
CONTINU
E…..
LIGHT
SCATTERING
Deflect in light
pathway due to
presence of large and
irregular substance .
Give idea about size
and internal complexity.
SSC – Change in RI .
CONTINUE…….
FLUORESCENCE
Fluorescent marker
used.
General fluorescence
principle.
Propidium iodide
(D) ,
phospatidylserine
(AC)
GENERAL STEPS
INVOLVED
SAMPLE INTRODUCE INTO LASER
PREPARATION SAMPLE CHAMBER DETECTION
DATA SIGNAL
ANALYSIS PROCESSING
INSTRUMENTATION
COMPONENTS OF FLOW
CYTOMETER
OPTICAL SIGNALLING
FLUIDIC SYSTEM SYSTEM
SAMPLE INJECTION DETECTING PROCESSING
LASER
PORT
SHEATH FLUID LENSES CONVERT
PHOTODIOD LIGHT INTO
E (FSC) VOLTAGE
OPTICAL FILTER &
FLOW CELL PMT(SSC) PULSE
DICHROIC MIRROR
HISTOGRA
M & DOT
PLOT
SAMPLE PREPARATION
SAMPLE COLLECTION
BLOOD SAMPLE – collected in EDTA or heparin
TISSUE SAMPLE – Sterile buffer or culture media
CELL CULTURE SAMPLE – Harvest adherent
CELL SUSPENSION PREPARATION
SOLID TISSUE – mechanical dissociation (mincing / sieving) or
enzymatic digestion
BLOOD OR BONE MARROW – Perform RBC lysis using NH4Cl lysis buffer
FILTER THE SUSPENSION – using 40-80 nylon mesh size
CONTINUE……
VIABILITY CHECK
First go for cell count
(haemocytometer)
CHECK VIABILTY – TRYPAN BLUE
OR PROPIDIUM IODIDE
FIXATION
FOR IC ANTIGEN - 70% ETHANOL
& 4% PARAFORMALDEHYDE
PERMEABILIZATION
SAPONIN TRITON X - 100
CELL PREPARATION CONTINUE………..
STAINING PROCEDURE FINAL
• Addition of PREPARATION
fluorochrome • SUSPEND
conjugated antibodies stained cell in
or dyes targeting FACS buffer
specific cellular markers. • Repeat
• Control used are filtration
UNSTAINED • Ice and dark
CONTROL place
SINGLE STAINED protection
CONTROL
ISOTYPE CTRL
ADVANTAGE DISADVANTA
S GES
• Measure multiple parameters • Expensive and complex
in same sample. instrumentation
• Rapid and efficient output. • Sample preparation should be
• Enables immunophenotyping accurate and specific.
to identify and quantify • Highly sensitive.
specific cell population • Limited morphological info.
• Distinguish apoptotic viable • Dead and dying cell may skew
and necrotic cell, result.
• Capable of detecting rare cell • Require regular calibaration .
family.
APPLICATION
PHARMA INDUSTRY REGENERETIVE MEDICINE CANCER RESEARCH
• TARGET IDENTIFY , • IDENTIFY & • HETEROGENIC
SCREENING,MOA CHARECTERIZE TUMOR PROFILING
• TOXICITY & SAFETY HEMATOPOIETIC • CANCER
EVALUATION , IMMUNOTHERAPY
• BIOMARKER MESENCHYMAL , EFFECTIVENESS EX-
DISCOVERY INDUCED CAR T cell
PLEURIPOTENT
STEM CELL
• PURIFY
STEMCELL
APPLICATION
MICROBIOLOGY OTHERS
APPLICATION
MICROBIOLOGY OTHERS
• DETECT COUNT & • QC IN
IDENTIFY BACTERIA BIOMANUFACTURING
YEAST &OTHER LIKE PROTEIN
MICROBES EXPRESSION
• MICROBIAL • AGRICULTURAL USE
COMMUNITY LIKE PLANT PROTEIN
DYNAMICS & AND CELL ANALYSIS
ANTIBIOTIC
RESISTANCE.
REFERENCE-
o[Link]
o[Link]
cell-analysis-learning-center/molecular-probes-school-of-fluorescen
ce/flow-cytometry-basics/flow-cytometry-fundamentals/how-flow-c
[Link]
o[Link]
pics/non-
[Link]
o[Link]
[Link]
o[Link]
nd-journals/bioprobes-journal-of-cell-biology-applications/bioprobes
-79/[Link]