really shitty biotech review for flow cytometry part 2 (rhyme for the mixtape) Flashcards
Monocytes originate in the _____ ______ from a rapidly growing pool of myeloid progenitor cells called __________.
1 - bone marrow
2 - promyelocytes
Monocytes are a type of ________ (WBC) that constitute the basic cellular components of the ________ blood in which they circulate in and make up between ___ and ___ % of the total WBCs in the body.
1 - leukocyte
2 - peripheral
3 - 5;10
After a short _______ ______ of 1-3 days, they then circulate briefly in the blood and migrate into the _______ where they assume their ultimate role as ________.
1 - developmental period
2 - tissues
3 - macrophages
_____ - ______ macrophages on the other hand, reside in the the tissue and are dedicated to fulfilling tissue specific functions including?
1 - Tissue resident
Functions include: clearance of cellular debris, play a central role in immune surveillance and the inflammatory response.
Macrophages respond in two primary ways. These are?
chemoattraction and adhesion.
What is chemoattraction?
When macrophages in the affected area release cytokines. This in turn causes the endothelial cells near the area, to express cellular adhesion molecules resulting in the localization of circulating leukocytes to the site of injury or infection.
To sum up:
macrophages release cytokines –> causes endothelial cells to express cellular adhesion molecules -> result is localization of circulating leukocytes to site of injury/infection
What are examples of cytokines?
IL-1 and TNF-alpha
What are examples of cellular adhesion molecules?
P- and E-selectin
What are endothelial cells?
cells that line blood vessels
What is the mechanism of adhesion?
Macrophages release chemokines (oh yeah thx scientists, put chemokines with adhesion rather than chemoattraction…) –> causes surface integrin molecules on the endothelial cells to kick into overdrive (definitely going in the lab report) going into a high-affinity state. –> causes immobilization of leukocytes to that area (Since integrins become tightly bound to complementary receptors on the endothelial cells) –> they then enter the tissues as an influx in order to differentiate and replenish tissue-specific macrophages
The differentiation process is due to what?
Result of signals from locally produced factors such as M-CSF, which prepares the cells to actively participate in teh inflammatory and innate immune response.
Upon differentiation, the cells lose their ability to _______ leading to enhanced ___________ activity enabling them to _______ and _______ (or _________) foreign material, dead cells, etc.
1 - replicate
2 - antibacterial
3 - engulf and digest or phagocytose
M-CSF stands for?
Macrophage - colony stimulating factor
What are examples of integrin molecules?
VCAM-1 and alpha4beta1
The human monocytic leukemia cell line, ____, can be induced to differentiate into ________ by treatment with ___ or _____.
1 - THP-1
2 - macrophages
3 - PAM or Vit-D3
Primary tissue macrophages are not readily expandable in _____ systems.
in-vivo
What does not expandable mean?
cannot grow them easily in large quantities
The _________ state and _________ of this cell line however can vary based on the type of _________ and _______ used to induce differentiation and can therefore only moderately predict the behaviour of typical ________ ________ in living systems.
1 - differentiation state 2 - phenotype 3 - treatment 4 - duration 5 - primary macrophages
Differentiation of monocytic cell lines into highly differentiated macrophages is a primary focus in order to closely resemble what?
MDM - monocyte derived macrophages
Further culture of ____ cells following treatment with ____ in regular _____, drives cells more towards the differentiation states of typical ____.
1 - THM-1
2 - PAM
3 - media
4 - MDMs
The expansion of the ________ is central to the function of differentiated _________ as this enhances their ________ capacity allowing for more cellular debris and foreign microbes to be cleared from the tissue.
1 - cytoplasm
2 - macrophages
3 - phagocytic
Further analysis by flow cytometry generally detects an observable increase in _____ _____ or _____/________.
1 - side scatter
2 - granularity/density
____ treatment also leads to a more mature ________ resulting in a slower rate of ________, higher levels of _______, higher rate of _________ in addition to an increased cell ______ expression of ____ and _____.
1 - PMA 2 - phenotypes 3 - proliferation 4 - phagocytosis 5 - adherence 6 - surface 7 - Cd14 and CD11b
Classically, an increase in _____ expression has been widely used as a factor to identify _______ of monocytes to macrophages however, more recently, scientists have discovered that the _____ culture conditions (______ and _______ length) of ____ monocytes, prior to any stimuli (such as ____ treatment), is a critical factor that can influence the _______ response and ____ of macrophages.
1 - CD14 2 - differentiation 3 - basal 4 - confluence and culture length 5 - THP-1 6 - PMA 7 - differentiated 8 - state
What is another well used approach for the classification and identification of macrophages?
Autofluorescence.
The full biological mechanism behind the resultant autofluorescence is not yet fully understood however, it has been suggested that it could be derived as a result of what?
increased complexity in the cells, flavins and/or cellular lipids
In this lab you will use ____ ______ as a means to examine the characteristic differences between ________ and _________ in ____ treated ____ cells.
1- flow cytometry
2 - monocytes and macrophages
3 - PMA treated THP-1
We will be analyzing the _____ light ____ and ____ _____ plots to identify features and differences in ____ and _______ of monocytes compared to macrophages.
1 - forward and side scatter
2 - size and granularity
How can autofluorescence be measured?
by the FL1 channel
The lysosome concentration should increase with _______ due to an increase in ______ volume, measured by the ___ channel.
1 - differentiation (monocytes –> macrophages)
2 - cytoplasm
3 - FL3
The different expression of cell-surface antibodies is measured by what?
1 - CD14-FITC via the FL1 channel
2 - CD4-Texas red via the FL3 channel
The phagocytic capacity of THP-1 cells should also _____ with differentiation due to an overall increase in ____ _____ and ______ and will be monitored through the ___ channel.
1 - increase
2 - size and volume
3 - FL3
