Biology Reference
In-Depth Information
21.4
ANALYSIS
For each cell replica, groups of gold particles are counted and categorized according
to the number of gold particles. Gold counts and the size distribution of observed
clusters are written down for each individual cell in a prepared table. When analyzing
replicas of cells labeled with antibodies specific for the TCR, we consider that gold
particles are part of the same cluster when they are adjacent or when the distance
between two particles is less than 10 nm (i.e., the diameter of a single gold particle),
taking into account that the diameter of the TCR is around 10 nm (
Arechaga et al.,
2010
) and that the labeling efficiency is low. As a starting point, we record the gold
particle distribution of 10 cell replicas of each condition (background controls
and experimental groups) and determine statistical significance of differences
(e.g., using two-tailed
T
-tests and chi-square tests). Automated data collection
methods and computational methods to determine cell surface receptor distribution
have been described (
Zhang et al., 2006
) but require careful filtering of the images to
discriminate gold particles from small holes and other irregularities that cause
electron-dense areas.
21.5
CONSIDERATIONS
21.5.1
Safety precautions
Use a fume hood and wear gloves and eye protection when working with PFA and
glutaraldehyde. Dispose of waste according to institutional guidelines. Wear eye pro-
tection and cryogloves when handling liquid nitrogen. Extreme care must be taken to
eliminate any possibility of explosion hazard when working with flammable gases.
The liquefied cryogens should be removed from the unit on completion of the freez-
ing work and disposed of after each experiment by evaporation within the fume hood
(ethane) or burned in a specific burner (propane).
21.5.2
Starting number of cells
The starting numbers provided are based on our experience with naive and activated
primary B and T cells and B- and T-cell lines. For other cell types grown in suspen-
sion, this may have to be adjusted empirically. Unlabeled, PFA-fixed cells can be
adhered to freshly cleaved and poly-
L
-lysine-treated micas, and upon glutaraldehyde
fixation and washing, the mica sheets can be observed under a phase-contrast light
microscope. Cells should not be layered on top of each other, as this complicates the
ability to discern individual cells. Too few cells on the mica, especially if they are
very round cells (such as naive lymphocytes), may lead to very high surface tension
around the replica of the actual cells, causing the cell replica to pop out. A cell den-
sity resulting in cells spaced at distances of approximately one cell diameter should
avoid these problems. Adherent cells could in principle be grown to confluency, but
