ADD interior quantification section
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@ -803,7 +803,7 @@ adding \gls{fitcbt} to the \gls{stp}-coated \glspl{dms}, resuspending in
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staining with \product{\anti{\gls{igg}}-\gls{fitc}}{\bl}{406001}, incubating for
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\SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope.
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\subsection{t cell culture}
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\subsection{t cell culture}\label{sec:tcellculture}
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% TODO verify countess product number
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Cryopreserved primary human T cells were either obtained as sorted
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@ -838,7 +838,24 @@ Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul}
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\product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and
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imaging on a spinning disk confocal microscope.
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% METHOD mtt staining and interior quantification
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\subsection{Quantifying cells on DMS interior}
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% TODO add a product number to MTT (if I can find it)
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Cells were stained and visualized using \gls{mtt}. \glspl{dms} with attached and
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loosely attached cells were sampled as desired and filtered through a
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\SI{40}{\um} cell strainer. While still in the cell strainer, \glspl{dms} were
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washed twice with \gls{pbs} and then dried by pulling liquid through the bottom
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of the cell strainer via a micropipette and dabbing with a KimWipe. \glspl{dms}
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were transferred to a 24 well plate with \SI{400}{\ul} media. \SI{40}{\ul}
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\gls{mtt} was added to each well and allowed to incubate for \SI{3}{\hour},
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after which \glspl{dms} with cell were visualized via a brightfield microscope.
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To quantify cells on the interior of \glspl{dms}, cells and \glspl{dms} were
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isolated analogously to those for the \gls{mtt} stain up until the drying step.
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Cells were then transferred to a tube containing \SI{400}{\ul} at
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\SI{5}{\mg\per\ml} dispase solution. \glspl{dms} were incubated and rotated for
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\SI{45}{\minute} at \SI{37}{\degreeCelsius}, after which cells were counted as
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already described in \cref{sec:tcellculture}.
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\subsection{quantification of apoptosis using Annexin-V}
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