Exp15 : Analysis of Lymphocytes Flashcards
1
Q
New experimental approaches are essential in elucidating the mechanisms & functional basis of immune dysregulation in px with:
A
- primary/congenital immunodeficiency
- secondary/acquired infection immunodeficiency
- autoimmune disorders
2
Q
Isolated lymphocytes can also be subjected to the following tests:
A
- HLA Typing
- Lymphocytotoxicity Assay
3
Q
Natural or Acquired
Inflammatory repose
A
Natural
4
Q
Natural or Acquired
Non specific/Generic response
A
Natural
5
Q
Natural or Acquired
- Complement
- NK cells
A
Natural
6
Q
Natural or Acquired
Doesn’t have an immunologic memory
A
Natural
7
Q
Natural or Acquired
Antibody production/secretion
A
Acquired
8
Q
Natural or Acquired
Specific (tailor fit) & has immunologic memory
A
Acquired
9
Q
Natural or Acquired
Major and principal cells: T and B Lymphocytes
A
Acquired
10
Q
Type of Acquired Immunity
Targets extracellular pathogens
A
Humoral
11
Q
Type of Acquired Immunity
Antibody is produced by plasma cells (specialized B cells)
A
Humoral
12
Q
Plasma cells (special Ed B cells) can function as:
A
Opsonins
13
Q
It can neutralize foreign antigens by coating it, thereby allowing macrophages to identify them
= Making them more susceptible to phagocytosis
A
Opsonins
14
Q
Type of Acquired Immunity
Target intracellular pathogens
A
Cell-mediated
15
Q
Type of Acquired Immunity
Primarily T cells
A
Cell-mediated
16
Q
It is the assessment of proliferative capacity of lymphocytes
A
Mitogen-Induced Blastogenesis
17
Q
It measures the rate of incorporation of the radiolabeled DNA nucleosides
A
Mitogen-Induced blastogenesis
18
Q
Mitogen-induced blastogenesis uses prolonged ___________ cultures with ____
A
peripheral blood mononuclear cell (PBMC) cultures with mitogens.
19
Q
Mitogens induce ______ which then triggers the proliferation and replication of B and T cells
A
Blastogenesis
20
Q
T or B cells Mitogens
Pokeweed mitogen
A
B cell mitogens
21
Q
T or B cells Mitogens
Staphylococcal protein A
A
B cell mitogens
22
Q
T or B cells Mitogens
Phytohemagglutinin
A
T cell mitogens
23
Q
T or B cells Mitogens
Concanavalin A
A
B cell mitogens
24
Q
It measures the response of the entire cell population
A
Standard MIB (Bulk Assay Method)
25
In the Bulk Assay method (standard MIB), (1) lymphocytes are **isolated** with ______________
Ficoll-Hypaque Density Gradient Centrifugation
26
In the Bulk Assay method (standard MIB), (2) lymphocytes **cultured** for several days with a _________
mitogen
27
In the Bulk Assay method (standard MIB), (3) ______________ (radiolabeled DNA nucleosides) is added to the media
Titrated thymidine
28
In the Bulk Assay method (standard MIB), (3) after adding titrated thymidine to the media, it is incubated for _____________
4 - 24 hrs
29
In the Bulk Assay method (standard MIB), (4) cells are harvested using **cell harvester** and are transferred to disks of ________
filter paper
30
In the Bulk Assay method (standard MIB), (5) disks are placed in _________ to measure **radioactive emissions**
Scintillation fluid (liquid scintillation counter)
31
In the Bulk Assay method (standard MIB), **radioactive emissions** are measured in ________________ to determine the number of **proliferated lymphocytes**
liquid scintillation counter
32
Cell isolation techniques are based on:
Physical factors:
- Density
- Size
- Differential **migration of blood components** during **centrifugation**
33
- **15-22** μm in diameter
- **kidney** shaped, **irregular nucleus**
- **light blue-grey** cytoplasm
- fine dust-like granules
- **Vacuolation** may be observed
- Differentiates into **tissue macrophages**
- larger than **lymohocytes**
Monocyte
34
- **8-12 μm / 12-16 μm** in diameter
- Nucleus may vary based on size and maturity
- have **rounder nucleus**
- Function in cellular defense **against viral infections**
- scanty cytoplasm
- can be T or B cells
Lymphocytes
35
**Heparinized blood** diluted in Hank’s balanced salt solution (HBSS)/ Phosphate Buffered Saline (PBS)
36
Cells with **lower density** than the medium are expected to be _________ the density gradient
above
37
**Highly dense** blood components and medium are expected to be ____________ the density gradient
below
38
In the isolation & identification of PBMC, the principle is **Heparinized blood** diluted in _______________/ **Phosphate Buffered Saline (PBS)**
Hank’s balanced salt solution (HBSS)
39
In the isolation & identification of PBMC, the principle is **Heparinized blood** diluted in **Hank’s balanced salt solution (HBSS)**/**Phosphate Buffered Saline (PBS)** and is **overlaid** on _________ and centrifuged
Ficoll-Hypaque Solution
40
_________________ of the formed elements of blood during centrifugation leads to the formation of layers containing different cell types
Differential migration
41
Interface of Plasma Layer and Ficoll-Hypaque solution contains the:
- lymphocytes
- other slow-sedimenting platelets & monocytes
42
The **lymphocytes** and **platelets** are **separated** from the other formed elements of the blood as they have ___________density compared to the **Ficoll-Hypaque solution**
less density
43
The **lymphocytes** and **platelets** are **separated** from the other formed elements of the blood as they have a ______________ than **granulocytes & RBCS**
Lower specific gravity
44
A high-molecular-weight **sucrose polymers** that **do not penetrate** biological membranes
Ficoll (Ficoll-Hypaque Medium)
45
**Separation method** using **Ficoll** is based on the ___________
lower density of nucleated blood cells
46
The ficoll (ficoll-hypaque medium) uses a _____________ to **separate** the cells of **similar density** from other blood constituents
density gradient medium
47
The conical tubes used in isolation of PBMCa has a volume capacity of
10 mL vol capacity
48
The preferred material for tube for isolation of PBMCs to prevent or **minimize adherence** of lymphocytes & monocytes on the surface of the test tubes are
- plastic tubes
- siliconized tubes
49
**Mononuclear cells** tend to adhere to _______
Glass
50
What type of centrifuge is used for isolating PBMCs
Swing tyoe
51
The centrifuge is refrigerated at _________ in order to maintain the temperature
18 - 20 C
52
For the isolation of PBMCs, what is the balanced salt solution used
Hank’s phosphate buffered saline
53
For the isolation of PBMCs, what is the density gradient solution used
Ficoll-Hypaque
54
The ficoll-hypaque density gradient solution used for isolation of PBMCs is stored at ________
18 C to 20 C
55
The spx of choice for isolation of PBMCs is a freshly drawn (less than ____ hrs) venous blood mixed with an anticoagulant
Less than 8 hrs
56
The anticoagulant that can be used for the spx of choice in isolation of PBMCs are
- EDTA
- ACD (acid-citrate-dextrose)
- Heparin
- CPD (citrate-phosphate-dextrose)
57
Another spx of choice for isolation of PBMCs besides anticoagulated is whole blood that is ______
Defibrinated
58
This process for spx of choice prevents the blood from clotting by **removing the fibrin**
Defibrination
59
It is a technique for defibrination but is no longer being utilized
Glass beads
60
In the dilution of blood spx, the diluent used is _________
Hank’s phosphate buffered saline
61
During the collection or aftee recovering of lymphocytes, this reagent is added to resuspend
Suspending agent
62
During the process of diluting the blood spx, this washing solution is added to **remove the interfering substances** or unwanted components.
Hank’s Phosphate Buffered saline
63
If HPBS is not available, what else’s can be used as an alternative washing solution?
Normal Saline Solution (NSS)
64
In diluting the blood spx, sterile centrifuge, prepare a _________ Dilution (1:1 Ratio) the blood sample
1:2 dilution
65
To mix the blood and buffer solution (HPBS), we must
- gently inverting
- drawing the mixture in & out of the pipette
66
What is the density of the density gradient medium Ficoll-Hypaque medium
1.077 g/mL
67
What are the sterile solution components of Ficoll-Hypaque
- polysucrose
- sodium diatrizoate
- calcium disodium ETDA
68
T or F
After adding the diluted blood, we must mix it thoroughly with the FHS in the tube
False ! (Must be 2 different layers)
69
After adding the diluted blood together with FHS in a tube, it is centrifuged in at 300x g at 20C for 30-40 mins using a _______
Swing-out bucket rotor (swing type centrifuge)
70
What are the layers formed after the first centrifugation (from top to bottom)
1. Plasma diluted in HPBS
2. Cell ring
3. FHS (histopaque)
4. Buffy coat
5. RBC Sediment / Packed RBC
71
What layer (after centrifugation) contains the mononuclear cells (lymphocytes), monocytes, & platelets
Cell ring
72
What layer (after contribution) contains the dense/big WBCs (large monocytes & granulocytes)
Buffy coat
73
T or F
Cells in the interphase must be aspirated without delay.
True
74
How many centrifugation must be done in isolating PBMCs
2 times
75
What do we use to wash the cell isolates?
Hank’s balanced salt solution
76
What do we do to render the PBMC sediments free of platelet
lower centrifugation speed at 60x-100x g for 10 mins at 18C to 20C.
77
What are the layers after the second centrifugation (from top to bottom)
1. Supernatant (platelets & Hank’s PBS)
2. Lymphocytes pellet
78
In the washing of the cell isolate, after two centrifugation, we dilute the cell pellet in ____ mL of HBSS
6-8 mL
79
What can we do to prevent the drying out of the cell pellets
Resuspend in Hank’s PBS
80
It purifies different leukocyte subsets such as T- and B-cells, even stem cells
Nylon wool purification
81
The nylon wool purification method uses ________ which have **not been specifically coated with ligand**
fiber
82
It relies on the affinity of certain lymphoid cells to bind to the nylon support
nylon wool purification
83
In the nylon wool purification, **small resting precursor** cells of _____________ adhere significantly to nylon wool columns
B-lymphocytes (has nylon wool receptor)
84
__________ can be easily washed off from nylon
T-lymphocytes
85
It is the lymphocyte that has presence of nylon wool receptor
B cell
86
What is the spx for nylon wool purification
Lymphocyte pellets of lymphocyte cell isolate
87
In the nylon wool purification, the **Dulbecco’s PBS** must contain
- 5% heat inactivated fetal calf serum (FCS)
- Hank’s balance salt solution (w 10% FCS)
88
In the separation of T & B-cell, the separating column consists of:
Syringe with stopcock
89
Nylon wool will not stimulate nor react with the lymphocytes, which means it is
Non-Antigenic
90
It is used to wash the nylon wool to moisten the wool and to enhance adherence properties of the nylon wool
Hank’s PBS with 10% FCS (fetal calf serum)
91
What do we use to collect the adherent B-cells from the column?
- RPMI-1640
- McCoy media
92
What are used to manually count the peripheral blood and nuclear cells or PBMC such as lymphocytes
Hematocytometer and Light microscopd
93
What type of hemocytometer is used for the ration of isolated T & B cells?
Improved Neubauer counting chamber
94
What is the diluent used for the enumeration of lymphocyte cell isolate?
3% acetic acid
95
What is the dye used to stain the lymphocyte pellets?
Trypan blue
96
How many minutes is the needed for the mixture to stand if the total nucleus count from the lymphocyte cell suspension is desired?
15 minutes (causes lysis and the release of its nucleus)
97
It has been included a supplemental procedure to determine the **number of viable cells present** in the **isolated lymphocyte suspensions**
Trypan blue dye exclusion test
98
It is one of the traditional methods used in assessing cell viability
Trypan blue dye exclusion test
99
It is the standard method used in academic research, laboratories and industrial biotechnology plants
Trypan blue dye exclusion test
100
In the Trypan blue dye exclusion test, live cells appear _________ as they have intact membranes and are not porous
Clear/transparent
101
In the Trypan blue dye exclusion test, dead cells appear _________ as they do not have intact membranes and take up the dye
Blur
102
Trypan blue dye exclusion test
Viable cell = ________
Non-Viable cell = ________
Viable cell = clear
Non-Viable cell = blue
103
What is the Trypan blue dye used in TB Exclusion test
Trypan blue (0.2% solution in PBS)
104
What is the formula for :
% of Viable cells
# of viable cells (unstained) / total # of cells
105
What is the formula for :
Number of cells (viable or non-viable)
(A+B+C+D / 4) x 10^4 x 2 x sample dilution
106
T or F
In a live cells, the certain portion appears colorless while the edges are stains since **Trypan blue can penetrate the cytoplasm**
True
107
This test is used to identify T cells
E-rosette assay
108
T cells have specific markers _________ on the surface, which will be the property used to identify T cells
CD2
109
What is the reagent used in E-rosette assay?
Sheep’s blood (receptor: CD58)
110
In the e-rosette assay, T cells would be surrounded with the sheep RBC in a rosette form since sheep RBC CD___ will attach to the CD___ of the T cells
CD58 <—> CD2
111
T or F
B cell isolate negative in E-rosette assay (since B cell has no CD2 marker)
True
