S1 [LAB]: Exp. 15: Analysis of Lymphocytes Flashcards
The assessment of proliferative capacity of lymphocytes
Mitogen-Induced Blastogenesis
Measures the rate of incorporation of the radiolabeled DNA nucleosides following prolonged incubation of the peripheral blood mononuclear cell (PBMC) cultures with mitogens
Mitogen-Induced Blastogenesis
In standard MIB (Bulk Assay Method), lymphocytes are isolated with ___ and cultured for several days with a mitogen
Ficoll-Hypaque Density Gradient Centrifugation
Mitogen used for T cell
Concavalin A
Mitogen used for B cell
Staphylococcal Protein A
Pokeweed Mitogen
A radiolabeled DNA nucleoside added to the media after culture for several days with mitogen
Titrated thymidine
Measures the response of the entire cell population
Bulk Assay Method
Size of monocytes
15-22um
Cell of the immune system that has a kidney bean-shaped nucleus
Monocyte
Cell of the immune system that has a light blue-grey cytoplasm containing fine dust-like granules
Monocyte
Monocytes differentiate into ___
Tissue macrophages
In monocytes, ___ may be observed in the cytoplasm
Vacuolation
Size of lymphocyte
8-12um/ 12-16um
T/F: The nucleus of the lymphocyte may vary based on size and maturity
True
The function of the lymphocyte include cellular defense against ___ infections
Viral
In PBMC, the cell isolation techniques are based on physical factors such as ___
Density and size
In PBMC, the cell isolation technique include the differential migration of blood components during ___
centrifugation
A high molecular weight sucrose polymers that do not penetrate biological membranes
Ficoll
The ___ and ___ should be at the interface of plasma layer and Ficoll-Hypaque solution
Lymphocytes
Other slow sedimenting platelets and monocytes
Separation method using Ficoll is based on the ___ of nucleated blood cells and uses a ___ medium to separate these cells of similar density from other blood constituents
Lower density
Density gradient
Specimen required for isolation of PBMCs
Freshly drawn anticoagulated blood (8 hrs old)
In the isolation of PBMC, a dilution of ___ blood sample is prepared by placing ___mL of anticoagulated blood on the tube and an equal volume of sterile HBSS/PBS
1:1
2mL
In the isolation of PBMCs, the total volume of the diluted blood sample should be ___
4mL
Centrifuge setting for the isolation of PBMCs
300x g at 20C for 30-40 mins
In the isolation of PBMCs, the upper layer is the ___
Plasma
Widely used due to its ease of use and cost effectiveness to purify different leukocyte subsets such as T and B cells, even stem cells
Nylon Wool Purification
Uses fibers which have not been specifically coated with ligand
Nylon wool purification
The principle of the isolation of PBMC include heparinized blood diluted in ___ or ___ overlaid on Ficoll-Hypaque solution and centrifuged
Hank’s balanced salt solution (HBSS)
Phosphate buffered saline (PBS)
The PBMC is formed in the layer between the ___ and ___
Histopaque
Medium
The mononuclear cells are in this layer during PBMC isolation
Whitish buffy coat
T/F: During PBMC isolation, cells in the interphase must be aspirated without delay
True
To render the PBMC sediments free of platelet, initially subject the PBMC sediments to what centrifugation settings?
Low speed (60-100x g), for 10 mins at 18-20C
Relies on the affinity of certain lymphoid cells to bind to the nylon support
Nylon wool purification
Culture medium for lymphocytes
RPMI-1640 (Roswell Park Memorial Institute)
Sample required for enumeration of isolated T and B cells
Lymphocyte pellets/ Lymphocyte cell isolate
___ and ___ are used to manually count the PBMCs
Hemacytometer
Light microscopy
Visual checking of PBMCs are aided by ___
Trypan Blue Exclusion Test
In counting the lymphocytes, one should wait at least ___ to allow the cells to settle before counting
30s
In counting the lymphocytes, these fields shall be used in the improved neubauer counting chambers
4 WBC Secondary Squares
The diluent for counting lymphocytes
3% acetic acid
The diluted lymphocyte is mixed well for ___ before manual counting
15mins
One of the traditional methods used in assessing cell viability
Trypan Blue Dye Exclusion Test
The standard method used in academic research laboratories and industrial biotechnology plants
Trypan Blue Dye Exclusion Test
In the visualization of PBMCs using Trypan Blue Dye Extraction Test, the dead cells appear ___ under the microscope
Blue
In the visualization of PBMCs using Trypan Blue Dye Extraction Test, living cells appear ___ under the microscope
Clear/ transparent
Uses of TB Exclusion/ Viability test
Cell counting
Differentiates viable versus non-viable cells
This lymphocyte isolation method uses monoclonal antibodies against T or B cell
Lymphokwik Assay
In Lymphokwik Assay, if the B cell should be isolated, the ___ should be used
T cell
This lymphocyte isolation method uses monoclonal antibodies specific for B cell or T cell; if B cell should be isolated, B cells should also be used
Magnetic Beads
Substances that induce proliferation of lymphocytes
Mitogens
In standard MIB, the addition of titrated thymidine is followed by incubation for ___
4-24 hours
T/F: Monocytes are more associated with mononuclear cells than lymphocytes
False (lymphocytes are more associated)
Used to dilute the heparinized blood sample during isolation of PBMCs
HBSS/PBS
Anticoagulant of choice for isolation of PBMCs
Heparin
The ___ is the layer containing the mononuclear cells when Ficoll-Hypaque solution is used
Cell ring
In the nylon wool purification technique, the ___ will be with the first eluate
T cell
In the nylon wool purification technique, the ___ will cling with the nylon wool
B cell
In the nylon wool purification technique, to allow the second eluate, nylon wool should be washed with ___
HBSS/PBS
