Practice #4 Flashcards
Flow cytometry/ FACS
Fluorescence activated cell sorting
Advantages of flow cytometry as oppose to microscope
- Objective 2. Large num of cells can be examined in 1 measurment 3. Automatic 4. Fast
The basic structure of flow cytometer
- Fluidics: cell flow and hydrodynamic focusing to the point of detection 2. Optics: Laser beam is concentrated and passes through one cell at the time on the other side the emitted light is detected by FSC (front scatter detector) and a series of SSC (side scatter detectors) 3. Electronics: transforming light signals to electric ones for FSC its photodiode and for SSC its PMT 4. Analysator unit: collection, storage and processing
Hydrodynamic focusing is done by
- The shape of the flow cell (funnel shape) 2. Sheath fluid (isitonic buffer) : flowing in laminar flow to the opposite direction of the sample stream
What are the functions of FSC and SSC?
FSC - Indicator of the cell size with direct proportionality SSC - Indicator for the complexity of the cell (granular or not)
What are the possible fluorescent markers used to tag the cell
Direct, indirect, biotin-avidin, intracellular molecules
Possible data imaging methods
- Histogram - one single parameter depending on cell count 2. Dot plot - 2 or more parameters
Applications of flow cytometry
- cell sorting 2. total cell count 3. detecting different cell populations in the sapmle 4. detection of the functional state of cells in the sample
Preparation of the sample for flow cytometry
- Fresh suspension (not coagulated blood) 2. Erythrolysis 3. Staining if necessary
Dot plot complexity vs. size
- Eosinophils
- Neutrophils
- Monocytes
- Lymphocytes and Basophils
Dot plot diagnosis
Left: Normal bone marrow smear
Right: Bone marrow leukemia
What happened between A and B?
In A there is more fluorescence proteins attached to each cell
Left shift
(intensity shift)
What happened between A and B?
In B the number of cells expressing CD3 is increased
What are the different quality check possibilities in flow cytometry?
- Technical:
setting the machine
Fluorescence compensation for molecules with lower intensity fluorescence
- Biological:
Unstained preparation
or
Unspecific binding to control isotope
Detection of autofluorescence
Markers for NKC
CD3-
CD56+
Markers for B cells
CD19+
Markers for B1 cells
CD5+
Markers for plasma cells
ic light chain +
CD19-
Markers for Th cells
CD3+
CD4+
Markers for T cells
CD3+
Markers for NKT cells
CD3+
CD56+
Markers for Tc cells
CD3+
CD8+
Markers for gamma/delta T cells
CD3+
gamma/delta TCR +
CD4-
CD8-
Markers for Treg cells
CD4+
CD25++
Foxp3+
Markers for Th1 cells
IFNgamma+
IL12+
TNFalpha+
TNFbeta+
Markers for Th2 cells
IL4+
IL5+
IL10+
IL13+
Markers for Th17 cells
IL17+
TGFbeta+
Markers for lymphoid progenitor cells
CD34+
cKit+
HLA-DR+
TdT+
Soluble cytokine detection by flow cytometry
A microbead with specific Ab against the cytokine is mixed with the sample
then a secondary Ab (with fluorescence dye) against the cytokine is added
Cell cycle analysis by flow cytometry
- membrane lysis
- DNA staininng by propium iodide
* elevation of the S is an indicatorfor malignancy
Phagocyte function analysis by flow cytometry
Phagosytosis of fluorescnece labled latex beads will release the dye and the intensity of fluorescence will be higher
(the x axis on the right the y axis on the left)
Cell sorting by flow cytometry
Two possibilities:
- Mechanic separation by laser interrogation
- Electrostatic separation
Acute myeloid leukemia markers
CD14+
HLD-DR+
CD13+
CD33+
icMPO+
Acute lymphoid leukemia markers
HLA-DR+
icTdT+
Non T all
CD19+ CD20+
(CALLA)
CD10+
T all
CD2+ CD3+ CD5+ CD7+
CD45
Present on all WBC except plasma cells
CD38
Typical activation marker of Th, Tc, B, NKC
Prognostic marker for leukemia
CD138
Highly expressed on all tumor cells
FCM in pharmacology
Pgp detection by immunophenotyping
Measure the time it takesto the cell to pump out the fluorescent labled drug
