From f91a944aa593a4ae7f5462ca113d4e99346aedfa Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 27 Jul 2021 16:37:58 -0400 Subject: [PATCH] ADD interior quantification section --- tex/thesis.tex | 21 +++++++++++++++++++-- 1 file changed, 19 insertions(+), 2 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 83b4744..371f943 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -803,7 +803,7 @@ adding \gls{fitcbt} to the \gls{stp}-coated \glspl{dms}, resuspending in staining with \product{\anti{\gls{igg}}-\gls{fitc}}{\bl}{406001}, incubating for \SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope. -\subsection{t cell culture} +\subsection{t cell culture}\label{sec:tcellculture} % TODO verify countess product number Cryopreserved primary human T cells were either obtained as sorted @@ -838,7 +838,24 @@ 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. -% METHOD mtt staining and interior quantification +\subsection{Quantifying cells on DMS interior} + +% TODO add a product number to MTT (if I can find it) +Cells were stained and visualized using \gls{mtt}. \glspl{dms} with attached and +loosely attached cells were sampled as desired and filtered through a +\SI{40}{\um} cell strainer. While still in the cell strainer, \glspl{dms} were +washed twice with \gls{pbs} and then dried by pulling liquid through the bottom +of the cell strainer via a micropipette and dabbing with a KimWipe. \glspl{dms} +were transferred to a 24 well plate with \SI{400}{\ul} media. \SI{40}{\ul} +\gls{mtt} was added to each well and allowed to incubate for \SI{3}{\hour}, +after which \glspl{dms} with cell were visualized via a brightfield microscope. + +To quantify cells on the interior of \glspl{dms}, cells and \glspl{dms} were +isolated analogously to those for the \gls{mtt} stain up until the drying step. +Cells were then transferred to a tube containing \SI{400}{\ul} at +\SI{5}{\mg\per\ml} dispase solution. \glspl{dms} were incubated and rotated for +\SI{45}{\minute} at \SI{37}{\degreeCelsius}, after which cells were counted as +already described in \cref{sec:tcellculture}. \subsection{quantification of apoptosis using Annexin-V}