Transcript CELL BIOLOGY TECHNIQUES

CELL BIOLOGY TECHNIQUES
Visualize cells - Microscopy
Organelles – Fractionation of
subcellular components
Culturing cells
Light Microscopy
Light Microscopy
• Resolution of 0.2µm
• Magnification – objective and projection lens
• Resolution
– D = 0.61λ/N sin α
Resolution is improved by using shorter
wavelengths or increasing either N or α.
BRIGHT FIELD PATH MICROSCOPY
Visualize unstained living cells
• Phase Contrast microscopy
– Thin layers of cells but not thick tissues
• Differential Interference contrast
– Suited for extremely small details and thick
objects
– Thin optical section through the object
Microscopy of Live cells
Fluorescence Microscopy
• Major Function: Localization of specific cellular
molecules – example proteins
• Major Advantages:
– Sensitivity:“glow” against dark background
– Specificity: immunofluorescence
– Cells may be fixed or living
• Fluorescent dyes or proteins (Flurochromes)
– flurochromes may be indirectly or directly associated
with the cellular molecule
– Multiple flurochromes may be used simultaneously
Absorb light at one
wavelength and
emit light at a
specific and longer
wavelength
HYDRA EXPRESSING GFP
Fluorescent
protein in live
cells
FIX
EMBED
SECTION
STAIN
Immunofluorescence Microscopy and
Specific Proteins
• Fluorescently tagged primary anti body
• Fluorescently tagged secondary antibody
• Fluorescently labelled antibody to tagged
proteins such as myc or FLAG
RAT INTESTINAL CELL WALL – GLUT 2
CONFOCAL AND DECONVOLUTION
MICROSCOPY
• This overcomes the limitations of
Fluorescence microscopy
– Blurrred images
– Thick specimens
REMOVES OUT OF FOCUS IMAGES
EXAMPLE OF IMAGE RECONSTRUCTED
AFTER DECONVOLUTION MICROSCOPY
ELECTRON
MICROSCOPY
• Transmission EM
– theoretically 0.005 nm; practically 0.1 nm –1 nm
(2000x better than LM)
– High – velocity electron beam passes through the
sample
– 50-100 nm thick sections
– 2-D sectional image – surface details are revelaed
– Subcellular organelles
• Scanning EM
– Resolution about 10 nm
– Secondary electrons released from the metal coated
unsectioned specimen
– 3-D surface image
GOLD PARTICLES COATED WITH PROTEIN A ARE
USED TO DETECT ANTIBODY BOUND TO PROTEIN
TEM IMAGE
CRYOELECTRON MICROSCOPY
• HYDRATED, UNFIXED AND UNSTAINED
SAMPLES
• SAMPLES ARE OBSERVED IN ITS NATIVE
HYDRATED STATE
• METHOD - AN AQUEOUS SUSPENSION OF
SAMPLE IS APLLIED ON A GRID AND HELP B Y
A SPECIAL MOUNT
• 5 nm RESOLUTION
SURFACE DETAILS BY METAL
SHADOWING
SEM OF EPITHELIUM LINING THE
INTESTINAL LUIMEN
PURIFICATION OF CELL ORGANELLES
• CELL DISRUPTION
• SEPARATION OF DIFFERENT ORGANELLES
USING CENTRIFUGATION
• PREPARATION OF PURIFIED ORGANELLES
USING SPECIFIC ANTIBODIES
BREAKING OPEN PLASMA
MEMBRANES IN CELLS
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CELLS ARE SUSPENDED IN ISOTONIC SUCROSE
SONICATION
HOMOGENIZATION
CELLS IN HYPOTONIC SOLUTION – RUPTURE
OF CELL MEMBRANES
SEPERATING ORGANELLES
• DIFFERENTIAL CENTRIFUGATION
• DENSITY GRADIENT CENTRIFUGATION
DENSITY GRADIENT CENTRIFUGATION
ANTIBODIES ARE USED TO MAKE
HIGHLY PURIFIED ORGANELLES
CELL SORTER – FLOW CYTOMETRY
CELL CULTURE REQUIREMENTS
• SOLID MEDIA
– Specially coated plastic dishes or flasks (CAMs’)
– Agar as the medium
GROWTH MEDIA
Rich in nutrients- amino acids, vitamins, salts fatty
acids, glucose, serum provides the different
growth factors,
TYPES OF CULTURED CELLS
• PRIMARY CELL CULTURES – DIFFERENTIATE IN
CELL CULTURE
• CELL STRAIN – ALSO HAVE A FINITE LIFE SPAN
(FROM A PRIMARY CULTURE)
• CELL LINE - INDEFINITE LIFE SPAN
PRIMARY CULTURES
STAGES IN CELL CULTURE
DIFFERNTIATION OF A CELL LINE –
C2C12 IN CULTURE
HOMEWORK-1
• CHAPTER 9
– REVIEW CONCEPTS QUESTIONS -2,5,7,9
– ANALYZE THE DATA
DUE NEXT WEEK IN THE WORKSHOPS