MCB 104 Exam 3#1 Questions with correct answers
cell counts - ✔✔required in numerous research experiments, whether using cells
grown in culture of obtaining cells from tissue
hemacytometer - ✔✔thick glass slide with etched lines for counting various blood
cells
one "square" is made up of 16 mini squares (9 squares per slide)
cell viability - ✔✔assessment of the percentage of live cells
viability estimate is done with an exclusion dye, such as trypan blue
diluting cells in trypan blue is often done in conjunction with a cell count as a
standard check to make sure you are using live cells for your assay
# of cells in square x 10^4
1:10 dilution in trypan blue, so multiply again by 10
then multiply by how many mls
in vitro - ✔✔experiments performed on cells in culture
in vivo - ✔✔experiments that are done in live animals
,primary cell lines - ✔✔have a finite number of cell divisions
transformed cell lines - ✔✔permanently growing cells with an infinite number of
cell divisions
cell culture conditions - ✔✔optimal growth conditions in incubators vary with
different cell types
standard for animal cells:
- 37ºC
- 5% CO2
- high humidity
cell culture media - ✔✔liquid medium that is reminiscent of the lymph fluid that
bathes cells
most media are balanced salt solutions with standard additives: amino acids,
vitamins, glucose
some media contain increased levels of nutrients
types of media: Eagles, MEM, DME, RPMI, Ham's F12, IMDM
common additives:
,1) pH indicator: most media contain buffering reagents to keep pH around 7-7.4
- phenol red --> red at 7.4, orange at 7, yellow at 6.5 and purple-red at 8
2) serum: most cell culture media have 2-20% fetal bovine serum added before
use
- serum = fluid that remains after blood is allowed to clot
- plasma is the blood fluid remaining after cells have been removed usually by
centrifugation (includes clotting factors because no clotting)
3) antibiotics
conditioned media contain more growth factors from growing cells
- cells usually like to grow in the company of other cells thus when cells are slow
growing or in low numbers then they require some form of conditioned media
- contain cytokines and hormones
antibodies/immunoglobulins - ✔✔can bind to literally anything
each B cell makes only one Ab with a specific antigen specificity
therefore, each antibody binds to only one epitope or antigenic determinant
polyclonal antibodies (polyclonal sera) - ✔✔in response to an invading pathogen,
you will activate many different B cells with a variety of antigen specificities to
many different proteins and different epitopes on these proteins
, B cell hybridoma - ✔✔fusion of a permanently growing (transformed) cell line
with a primary cell of choice
monoclonal antibodies are produced by B cell hybridomas which are the product
of a fusion between a B cell myeloma and a plasma B cell
you can make B cell hybridomas for monoclonal antibody production and T cell
hybridomas to test antigen reactivity to a specific T cell epitope
steps to making a monoclonal antibody - ✔✔1) priming or immunization: inject
mice 3-4 times with antigen usually combined with adjuvant in first
immunizations
- repeated injections of Ag will increase number of antibody producing B cells as
well as increase the affinity of the antibodies
- adjuvant: triggers a more vigorous adaptive immune response
- used Sigma adjuvant system (which consists of squalene and modified bacterial
products designed to provide enhanced immunogenicity)
- obtain serum from immunized mice then checked for antigen specific reactivity
by ELISA before the spleen cells are used in fusion
2) fusion: fuse myeloma cells with spleen cells from BALB/c mice immunized with
antigen
- myeloma cells are HGPRT-, normal spleen cells are HGPRT+
- add PEG to enhance fusion of myeloma cells with spleen cells (need to have
enough time but not too much time, as PEG can be toxic to cells)
- PEG: enhances membrane fusion by inducing the agglutination and cell-to-cell
contact, leading to subsequent cell fusion
cell counts - ✔✔required in numerous research experiments, whether using cells
grown in culture of obtaining cells from tissue
hemacytometer - ✔✔thick glass slide with etched lines for counting various blood
cells
one "square" is made up of 16 mini squares (9 squares per slide)
cell viability - ✔✔assessment of the percentage of live cells
viability estimate is done with an exclusion dye, such as trypan blue
diluting cells in trypan blue is often done in conjunction with a cell count as a
standard check to make sure you are using live cells for your assay
# of cells in square x 10^4
1:10 dilution in trypan blue, so multiply again by 10
then multiply by how many mls
in vitro - ✔✔experiments performed on cells in culture
in vivo - ✔✔experiments that are done in live animals
,primary cell lines - ✔✔have a finite number of cell divisions
transformed cell lines - ✔✔permanently growing cells with an infinite number of
cell divisions
cell culture conditions - ✔✔optimal growth conditions in incubators vary with
different cell types
standard for animal cells:
- 37ºC
- 5% CO2
- high humidity
cell culture media - ✔✔liquid medium that is reminiscent of the lymph fluid that
bathes cells
most media are balanced salt solutions with standard additives: amino acids,
vitamins, glucose
some media contain increased levels of nutrients
types of media: Eagles, MEM, DME, RPMI, Ham's F12, IMDM
common additives:
,1) pH indicator: most media contain buffering reagents to keep pH around 7-7.4
- phenol red --> red at 7.4, orange at 7, yellow at 6.5 and purple-red at 8
2) serum: most cell culture media have 2-20% fetal bovine serum added before
use
- serum = fluid that remains after blood is allowed to clot
- plasma is the blood fluid remaining after cells have been removed usually by
centrifugation (includes clotting factors because no clotting)
3) antibiotics
conditioned media contain more growth factors from growing cells
- cells usually like to grow in the company of other cells thus when cells are slow
growing or in low numbers then they require some form of conditioned media
- contain cytokines and hormones
antibodies/immunoglobulins - ✔✔can bind to literally anything
each B cell makes only one Ab with a specific antigen specificity
therefore, each antibody binds to only one epitope or antigenic determinant
polyclonal antibodies (polyclonal sera) - ✔✔in response to an invading pathogen,
you will activate many different B cells with a variety of antigen specificities to
many different proteins and different epitopes on these proteins
, B cell hybridoma - ✔✔fusion of a permanently growing (transformed) cell line
with a primary cell of choice
monoclonal antibodies are produced by B cell hybridomas which are the product
of a fusion between a B cell myeloma and a plasma B cell
you can make B cell hybridomas for monoclonal antibody production and T cell
hybridomas to test antigen reactivity to a specific T cell epitope
steps to making a monoclonal antibody - ✔✔1) priming or immunization: inject
mice 3-4 times with antigen usually combined with adjuvant in first
immunizations
- repeated injections of Ag will increase number of antibody producing B cells as
well as increase the affinity of the antibodies
- adjuvant: triggers a more vigorous adaptive immune response
- used Sigma adjuvant system (which consists of squalene and modified bacterial
products designed to provide enhanced immunogenicity)
- obtain serum from immunized mice then checked for antigen specific reactivity
by ELISA before the spleen cells are used in fusion
2) fusion: fuse myeloma cells with spleen cells from BALB/c mice immunized with
antigen
- myeloma cells are HGPRT-, normal spleen cells are HGPRT+
- add PEG to enhance fusion of myeloma cells with spleen cells (need to have
enough time but not too much time, as PEG can be toxic to cells)
- PEG: enhances membrane fusion by inducing the agglutination and cell-to-cell
contact, leading to subsequent cell fusion
