ADD methods to aim 2b

tex/thesis.tex

@@ -53,6 +53,8 @@
 \newacronym{pdms}{PDMS}{polydimethylsiloxane}
 \newacronym{dc}{DC}{dendritic cell}
 \newacronym{il2}{IL2}{interleukin 2}
+\newacronym{il15}{IL15}{interleukin 15}
+\newacronym{il15r}{IL15R}{interleukin 15 receptor}
 \newacronym{rhil2}{rhIL2}{recombinant human interleukin 2}
 \newacronym{apc}{APC}{antigen presenting cell}
 \newacronym{mhc}{MHC}{major histocompatibility complex}
@@ -104,7 +106,11 @@
 \newacronym{fcs}{FCS}{flow cytometry standard}
 \newacronym{ivis}{ivis}{in vivo imaging system}
 \newacronym{iacuc}{IACUC}{institutional animal care and use committee}
-
+\newacronym{hbss}{HBSS}{Hank's buffered saline solution}
+\newacronym{leaf}{LEAF}{low endotoxin, azide-free}
+\newacronym{cytof}{CyTOF}{cytometry time-of-flight}
+\newacronym{spade}{SPADE}{spanning-tree progression analysis of
+  density-normalized events}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 % SI units for uber nerds
@@ -2076,6 +2082,64 @@ provide these benefits.
 
 \section{introduction}
 \section{methods}
+
+\subsection{collagenase digestion}
+
+% TODO The concentration for the surface marker cleavage experiment was much
+% higher, if that matters
+\glspl{dms} were digested in active T cell cultures via addition of sterile
+\product{\gls{colb}}{Sigma}{11088807001} or
+\product{\gls{cold}}{Sigma}{11088858001}. Collagenase was dissolved in
+\product{\gls{hbss}}{Gibco}{14025-076} or
+\product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}.
+This solution was added to T cell cultures at a 1:1 ratio in place of plain
+media normally used to feed the cells during the regular media addition cycle at
+day 4. Cultures were then incubated as described in \cref{sec:tcellculture}, and
+the \glspl{dms} were verified to have been digested after \SI{24}{\hour}.
+
+\subsection{mass cytometry and clustering analysis}
+
+T cells were stained using a \product{34 \gls{cytof} marker
+  panel}{Fluidigm}{201322} and \product{cisplatin}{Fluidigm}{201064} which were
+used according to the manufacturer’s instructions. \numrange{2e6}{3e6} stained
+cells per group were analyzed on a Fluidigm Helios.
+
+% FIGURE add the spade gating diagram from the paper
+
+Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three
+representative \gls{fcs} files and running the \gls{spade} pipeline with k-means
+clustering (k = 100), arcsinh transformation with cofactor 5, density
+calculation neighborhood size of 5 and local density approximation factor of
+1.5, target density of 20000 cells, and outlier density cutoff of
+\SI{1}{\percent}. All markers in the \gls{cytof} panel were used in the analysis
+
+\subsection{integrin blocking experiments}
+
+To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were
+supplemented with \product{\anti{\gls{a2b1}}}{Sigma}{MAB1973Z} and
+\product{\anti{\gls{a2b2}}}{Sigma}{MAB1950Z} (both \gls{leaf}) at indicated
+concentrations and timepoints. T cells were grown as described in
+\cref{sec:tcellculture}.
+
+\gls{a2b1} and \gls{a2b2} were verified to be present on active T cell cultures
+by staining with \product{\anti{\gls{a2b1}}-\gls{apc}}{\bl}{328313} and
+\product{\anti{\gls{a2b2}}-\gls{fitc}}{\bl}{359305} on day 6 of culture and
+analyzing via a BD Accuri flow cytometer.
+
+\subsection{IL15 blocking experiments}
+
+To block the \gls{il15r}, we supplemented T cell
+cultures activated with \gls{dms} with either
+\product{\anti{\gls{il15r}}}{Rnd}{AF247} or \product{\gls{igg} isotype
+  control}{RnD}{AB-108-C} at the indicated timepoints and concentrations. T
+cells were grown as otherwise described in \cref{sec:tcellculture} with the
+exception that volumes were split by $\frac{1}{3}$ to keep the culture volume
+constant and minimize the amount of \gls{mab} required.
+
+To block soluble \gls{il15}, we supplemented analogously with
+\product{\anti{\gls{il15}}}{RnD}{EEP0419081} or \product{\gls{igg} isotype
+  control}{\bl}{B236633}.
+
 \section{results}
 
 \subsection{adding or removing DMSs alters expansion and phenotype}