By Frank J. Dixon (Ed.)
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Additional info for Advances in Immunology, Vol. 52
The number of voxels determines the minimal size of the particles to be analyzed (usually 3–5). Select the “connected components” algorithm to calculate the tracks for detected surfaces. Indicate the minimal duration for calculation. The resulting surface and track calculation should be similar to that observed in Fig. 2a. Select the MTOC and split it from the tracks. It connects several tracks at a time. At this point, the statistics calculated by the program contain all the tracks detected, even if they are branched (Fig.
2% TWEEN and 5% BSA. Blot membranes with primary antibodies (o/n at 4 °C) and peroxidase-conjugated corresponding secondary antibodies (30 min). 2% Tween at least three to four times each antibody. Detection of chemiluminescence signal may be performed with different imaging systems (see Note 12). 2 Preparation of poly-l-Lysine-Coated Surfaces 1. Mix anti-CD3 and anti-CD28 (3:1 ratio) antibodies in coating buffer. Add ICAM1-Fc (1 μg/ml). Pipette 50–100 μl of antibody mix per coverslip-bottom dish and incubate o/n at 4 °C.
Wash the cells twice with HBSS to exclude excess of antigen and resuspend in complete medium (4 × 10 6/ml). Use 100 μl for each time condition. For IF, preload B cells with a cell tracker such as CMAC (1 μM) in incomplete medium simultaneously with SEE or SEB/ HA peptide (Raji or Hom2, respectively); after 30 min of incubation, add FCS (5%) to the samples (see Note 11). Wash the cells twice with HBSS to exclude excess of antigen and resuspend in complete medium (3 × 106/ml). Use 50 μl for each coverslip preparation.