diff --git a/tex/thesis.tex b/tex/thesis.tex index 7139392..5d60c10 100644 --- a/tex/thesis.tex +++ b/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}