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ADD methods for apoptotic marker quantification

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Nathan Dwarshuis 2021-07-27 12:19:06 -04:00
parent b0e0f420eb
commit afbb5fadbd
1 changed files with 56 additions and 14 deletions
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tex/thesis.tex
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@ -88,8 +88,12 @@
\newacronym{colb}{COL-B}{collagenase B}
\newacronym{cold}{COL-D}{collagenase D}
\newacronym{tsne}{tSNE}{t-stochastic neighbor embedding}
\newacronym{anv}{ANX-V}{Annexin-V}
\newacronym{anv}{AXV}{Annexin-V}
\newacronym{pi}{PI}{propidium iodide}
\newacronym{rt}{RT}{room temperature}
\newacronym{cas37}{Cas3/7}{Caspase-3/7}
\newacronym{bcl2}{BCL-2}{B cell lymphoma 2}
\newacronym{tmb}{TMB}{3,3',5,5'-Tetramethylbenzidine}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% SI units for uber nerds
@ -103,7 +107,10 @@
\DeclareSIUnit\dms{DMS}
\DeclareSIUnit\cell{cells}
\DeclareSIUnit\ab{mAb}
\DeclareSIUnit\normal{N}
\DeclareSIUnit\molar{M}
\DeclareSIUnit\mM{\milli\molar}
\DeclareSIUnit\uM{\micro\molar}
\DeclareSIUnit\gforce{\times{} g}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
@ -710,7 +717,7 @@ carried out at \SI{20}{\mg\per\ml} carriers at room temperature and agitated
using an orbital shaker with a \SI{3}{\mm} orbit diameter. After autoclaving,
the microcarriers were washed using sterile \gls{pbs} three times in a 10:1
volume ratio. \product{\Gls{snb}}{\thermo}{21217} was dissolved at
approximately \SI{10}{\micro\molar} in sterile ultrapure water, and the true
approximately \SI{10}{\uM} in sterile ultrapure water, and the true
concentration was then determined using the \gls{haba} assay (see below).
\SI{5}{\ul\of{\ab}\per\mL} \gls{pbs} was added to carrier suspension and allowed
to react for \SI{60}{\minute} at \SI{700}{\rpm} of agitation. After the
@ -725,16 +732,17 @@ reaction volume.
To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\gls{stp}}{Jackson
Immunoresearch}{016-000-114} was added and allowed to react for
\SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, supernatant
was taken for the \gls{bca} assay, and the carriers were washed analogously to
the previous wash step to remove the biotin, except two washes were done and the
agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3
\catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass
ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along
with the \glspl{mab}, sterile \product{\gls{bsa}}{Sigma}{A9576} was added to a
final concentration of \SI{2}{\percent} in order to prevent non-specific binding
of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the
carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding,
supernatants were sampled to quantify remaining \gls{mab} concentration using an
was taken for the \product{\gls{bca} assay}{\thermo}{23225}, and the carriers
were washed analogously to the previous wash step to remove the biotin, except
two washes were done and the agitation time was \SI{30}{\minute}. Biotinylated
\glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904}
were combined in a 1:1 mass ratio and added to the carriers at
\SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile
\product{\gls{bsa}}{Sigma}{A9576} was added to a final concentration of
\SI{2}{\percent} in order to prevent non-specific binding of the antibodies to
the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
sampled to quantify remaining \gls{mab} concentration using an
\product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized
\glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing
step to remove excess \gls{stp}. They were washed once again in the cell culture
@ -767,7 +775,7 @@ extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
\SI{34000}{\per\cm\per\molar}.
\gls{stp} binding to the carriers was quantified indirectly using a
\product{\gls{bca} kit }{\thermo}{23227} according to the manufacturer’s
\product{\gls{bca} kit}{\thermo}{23227} according to the manufacturer’s
instructions, with the exception that the standard curve was made with known
concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at
\SI{592}{\nm} was quantified using a Biotek plate reader.
@ -826,6 +834,40 @@ Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul}
\product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and
imaging on a spinning disk confocal microscope.
\subsection{quantification of apoptosis using Annexin-V}
Apoptosis was quantified using \gls{anv} according to the manufacturer's
instructions. Briefly, cells were transferred to flow tubes and washed twice
with \gls{pbs} by adding \SI{3}{\ml} to each tube, centrifuging for
\SI{400}{\gforce}, and aspirating the liquid down to \SI{200}{\ul}. The cells
were analogously washed a third time with staining buffer (\SI{10}{\mM} HEPES,
\SI{140}{\mM} NaCl, \SI{2.5}{\mM} CaCl\textsubscript{2}) and aspirated down to a
final volume of \SI{100}{\ul}. Cells were stained in this volume with
\SI{1}{\ul} \product{\gls{anv}-\gls{fitc}}{\bl}{640906} and \SI{5}{\ul}
\product{\gls{pi}}{\thermo}{P3566} and incubated for \SI{15}{\minute} at gls{rt}
in the dark. After incubation, \SI{400}{\ul} staining buffer was added to each
tube. Each tube was then analyzed via flow cytometry.
\subsection{quantification of Caspase-3/7}
\Gls{cas37} was quantified using \product{CellEvent dye}{\thermo}{C10723}
according the manufacturer's instructions. Briefly, a 2X (\SI{8}{\mM}) working
solution of CellEvent dye was added to \SI{100}{\ul} cell suspension (at least
\num{5e4} cells) and incubated at \SI{37}{\degreeCelsius} for \SI{30}{\minute}.
After incubation, cells were immediately analyzed via flow cytometry.
\subsection{quantification of BCL-2}
\Gls{bcl2} was quantified using an \product{Human Total Bcl-2 DuoSet \gls{elisa}
kit}{Rnd Systems}{DYC827B-2} according to the manufacturer's instructions and
supplemented with \product{5X diluent buffer}{\bl}{421203}, \product{\gls{tmb}
substrate solution}{eBioscience}{00-4201-56}, and \SI{2}{\normal}
H\textsubscript{2}SO\textsubscript{4} stop solution made in house. Briefly,
cells were lysed using \product{10X lysis buffer}{Cell Signaling}{9803S}, and
the lysate was quantified for protein using a \product{\gls{bca}
assay}{\thermo}{23225} as directed. Standardized lysates were measured using
the \gls{elisa} kit as directed.
\subsection{chemotaxis assay}
% TODO not sure about the transwell product number
@ -882,7 +924,7 @@ plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
were removed from the retronectin plates using vigorous pipetting and
transferred to another 96 well plate wherein expansion continued.
% METHOD for apoptosis quantification
\subsection{statistical analysis}