ADD methods for apoptotic marker quantification

tex/thesis.tex

@@ -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
+was taken for the \product{\gls{bca} assay}{\thermo}{23225}, and the carriers
-the previous wash step to remove the biotin, except two washes were done and the
+were washed analogously to the previous wash step to remove the biotin, except
-agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3
+two washes were done and the agitation time was \SI{30}{\minute}. Biotinylated
-\catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass
+\glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904}
-ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along
+were combined in a 1:1 mass ratio and added to the carriers at
-with the \glspl{mab}, sterile \product{\gls{bsa}}{Sigma}{A9576} was added to a
+\SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile
-final concentration of \SI{2}{\percent} in order to prevent non-specific binding
+\product{\gls{bsa}}{Sigma}{A9576} was added to a final concentration of
-of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the
+\SI{2}{\percent} in order to prevent non-specific binding of the antibodies to
-carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding,
+the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
-supernatants were sampled to quantify remaining \gls{mab} concentration using an
+\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}