S1 [LAB]: Exp. 15: Analysis of Lymphocytes

Question 1

The assessment of proliferative capacity of lymphocytes

Answer 1

Mitogen-Induced Blastogenesis

Question 2

Measures the rate of incorporation of the radiolabeled DNA nucleosides following prolonged incubation of the peripheral blood mononuclear cell (PBMC) cultures with mitogens

Answer 2

Mitogen-Induced Blastogenesis

Question 3

In standard MIB (Bulk Assay Method), lymphocytes are isolated with ___ and cultured for several days with a mitogen

Answer 3

Ficoll-Hypaque Density Gradient Centrifugation

Question 4

Mitogen used for T cell

Answer 4

Concavalin A

Question 5

Mitogen used for B cell

Answer 5

Staphylococcal Protein A
Pokeweed Mitogen

Question 6

A radiolabeled DNA nucleoside added to the media after culture for several days with mitogen

Answer 6

Titrated thymidine

Question 7

Measures the response of the entire cell population

Answer 7

Bulk Assay Method

Question 8

Size of monocytes

Answer 8

15-22um

Question 9

Cell of the immune system that has a kidney bean-shaped nucleus

Answer 9

Monocyte

Question 10

Cell of the immune system that has a light blue-grey cytoplasm containing fine dust-like granules

Answer 10

Monocyte

Question 11

Monocytes differentiate into ___

Answer 11

Tissue macrophages

Question 12

In monocytes, ___ may be observed in the cytoplasm

Answer 12

Vacuolation

Question 13

Size of lymphocyte

Answer 13

8-12um/ 12-16um

Question 14

T/F: The nucleus of the lymphocyte may vary based on size and maturity

Answer 14

True

Question 15

The function of the lymphocyte include cellular defense against ___ infections

Answer 15

Viral

Question 16

In PBMC, the cell isolation techniques are based on physical factors such as ___

Answer 16

Density and size

Question 17

In PBMC, the cell isolation technique include the differential migration of blood components during ___

Answer 17

centrifugation

Question 18

A high molecular weight sucrose polymers that do not penetrate biological membranes

Answer 18

Ficoll

Question 19

The ___ and ___ should be at the interface of plasma layer and Ficoll-Hypaque solution

Answer 19

Lymphocytes
Other slow sedimenting platelets and monocytes

Question 20

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

Answer 20

Lower density
Density gradient

Question 21

Specimen required for isolation of PBMCs

Answer 21

Freshly drawn anticoagulated blood (8 hrs old)

Question 22

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

Answer 22

1:1
2mL

Question 23

In the isolation of PBMCs, the total volume of the diluted blood sample should be ___

Answer 23

4mL

Question 24

Centrifuge setting for the isolation of PBMCs

Answer 24

300x g at 20C for 30-40 mins

Question 25

In the isolation of PBMCs, the upper layer is the ___

Answer 25

Plasma

Question 26

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

Answer 26

Nylon Wool Purification

Question 27

Uses fibers which have not been specifically coated with ligand

Answer 27

Nylon wool purification

Question 28

The principle of the isolation of PBMC include heparinized blood diluted in ___ or ___ overlaid on Ficoll-Hypaque solution and centrifuged

Answer 28

Hank's balanced salt solution (HBSS) Phosphate buffered saline (PBS)

Question 29

The PBMC is formed in the layer between the ___ and ___

Answer 29

Histopaque Medium

Question 30

The mononuclear cells are in this layer during PBMC isolation

Answer 30

Whitish buffy coat

Question 31

T/F: During PBMC isolation, cells in the interphase must be aspirated without delay

Answer 31

True

Question 32

To render the PBMC sediments free of platelet, initially subject the PBMC sediments to what centrifugation settings?

Answer 32

Low speed (60-100x g), for 10 mins at 18-20C

Question 33

Relies on the affinity of certain lymphoid cells to bind to the nylon support

Answer 33

Nylon wool purification

Question 34

Culture medium for lymphocytes

Answer 34

RPMI-1640 (Roswell Park Memorial Institute)

Question 35

Sample required for enumeration of isolated T and B cells

Answer 35

Lymphocyte pellets/ Lymphocyte cell isolate

Question 36

___ and ___ are used to manually count the PBMCs

Answer 36

Hemacytometer Light microscopy

Question 37

Visual checking of PBMCs are aided by ___

Answer 37

Trypan Blue Exclusion Test

Question 38

In counting the lymphocytes, one should wait at least ___ to allow the cells to settle before counting

Answer 38

30s

Question 39

In counting the lymphocytes, these fields shall be used in the improved neubauer counting chambers

Answer 39

4 WBC Secondary Squares

Question 40

The diluent for counting lymphocytes

Answer 40

3% acetic acid

Question 41

The diluted lymphocyte is mixed well for ___ before manual counting

Answer 41

15mins

Question 42

One of the traditional methods used in assessing cell viability

Answer 42

Trypan Blue Dye Exclusion Test

Question 43

The standard method used in academic research laboratories and industrial biotechnology plants

Answer 43

Trypan Blue Dye Exclusion Test

Question 44

In the visualization of PBMCs using Trypan Blue Dye Extraction Test, the dead cells appear ___ under the microscope

Answer 44

Blue

Question 45

In the visualization of PBMCs using Trypan Blue Dye Extraction Test, living cells appear ___ under the microscope

Answer 45

Clear/ transparent

Question 46

Uses of TB Exclusion/ Viability test

Answer 46

Cell counting Differentiates viable versus non-viable cells

Question 47

This lymphocyte isolation method uses monoclonal antibodies against T or B cell

Answer 47

Lymphokwik Assay

Question 47

In Lymphokwik Assay, if the B cell should be isolated, the ___ should be used

Answer 47

T cell

Question 48

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

Answer 48

Magnetic Beads

Question 49

Substances that induce proliferation of lymphocytes

Answer 49

Mitogens

Question 50

In standard MIB, the addition of titrated thymidine is followed by incubation for ___

Answer 50

4-24 hours

Question 51

T/F: Monocytes are more associated with mononuclear cells than lymphocytes

Answer 51

False (lymphocytes are more associated)

Question 52

Used to dilute the heparinized blood sample during isolation of PBMCs

Answer 52

HBSS/PBS

Question 53

Anticoagulant of choice for isolation of PBMCs

Answer 53

Heparin

Question 54

The ___ is the layer containing the mononuclear cells when Ficoll-Hypaque solution is used

Answer 54

Cell ring

Question 55

In the nylon wool purification technique, the ___ will be with the first eluate

Answer 55

T cell

Question 56

In the nylon wool purification technique, the ___ will cling with the nylon wool

Answer 56

B cell

Question 57

In the nylon wool purification technique, to allow the second eluate, nylon wool should be washed with ___

Answer 57

HBSS/PBS