From afbb5fadbd9436a54479d12ac0356fdbb8617c2e Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 27 Jul 2021 12:19:06 -0400 Subject: [PATCH] ADD methods for apoptotic marker quantification --- tex/thesis.tex | 70 ++++++++++++++++++++++++++++++++++++++++---------- 1 file changed, 56 insertions(+), 14 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 428a760..b230661 100644 --- a/tex/thesis.tex +++ b/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 -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}