Inside our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells brought on specifically via their cognate antigen presented by MHCII. with at the half amplitude for and < 10?2; ***, < 10?3) (n = 20, 10, 10, and 7 cells from left to right). (b) Median Fura-2 ratio of activating T-cells for given conditions. Shaded regions were decided via bootstrapping. Cell spreading on activating surfaces is considered as a second hallmark for early T-cell signaling [18,20,21]. We investigated the formation of close contacts between the T-cell surface and functionalized SLBs using TIRF microscopy of the TCR specifically labeled using H57-AF647. Immediately upon contacting the surface, T-cells were observed to form individual, small, well-isolated, and highly dynamic contacts with the SLB, as had been described previously . After a few minutes, cells responded with a sudden increase in contact area (Physique 3a). For quantitative comparison, we define here the time-point of an increase in calcium or contact area as the time-point of the half-maximal effect (see Materials Hs.76067 and Methods), yielding concurrence of contact formation and calcium signal (Physique 3b). For Mirin better visualization of the correlation in the kinetics between calcium signal and contact area, we plotted the two values in a parametric plot, with time being the parameter (Physique 3c and Physique S2). Interestingly, while there were instances of low contact area at high Fura-2 ratios, we did not observe the opposite, i.e., high contact area at low Fura-2 ratio, in line with elevated calcium levels preceding  or even representing a precondition for T-cell adhesion . Open in a separate window Physique 3 Analysis of the T-cell contact area. (a) Median contact area as function of time. Before averaging, the time axis of each data set was shifted by the half-maximal time of the calcium signal. Shaded areas represent 95% confidence intervals. (b) Lag between half maximal area increase and calcium signal. Data are shown as Whisker box plots indicating the interquartile range (box), median (line), and the individual data points corresponding to single cell (circles) for high I-Ek/MCC + ICAM-1 + B7-1 (blue, n = 20 cells) and high I-Ek/MCC + B7-1 (orange, n = 10 cells). (c) Contact area of individual T-cells was plotted against the corresponding Fura-2 signal in a parametric plot, where time is the parameter. For each cell, we included 37 data points recorded in the time interval, ranging from 50 s before to 100 s after the calcium increase. Colors indicate density of data points. Data are shown for T-cells interacting with SLBs featuring ICAM-1, B7-1 and high I-Ek/MCC (n = 20 cells), B7-1 and high I-Ek/MCC (n = 10 cells), and low I-Ek/MCC (n = 10 cells) (ns, > 0.05). Delivery of single T-cells via the AFM cantilever allowed us to probe the conversation of T-cells with SLBs that were less potent for activation. Omitting ICAM-1 reduced the average area per cell after adhesion (Physique 3a), without any observable effect on the magnitude and kinetics of the calcium signal (Physique 2a and Physique 3b). We only observed an approximately 50% reduction of the number of activated cells (Physique S3). However, reducing the stimulus further by omitting both ICAM-1 and B7-1 and reducing the I-Ek/MCC surface density to 5 molecules per m2 yielded strong effects around the response. First, and in line with a previous report , the calcium response was delayed (Physique 2a) and lower in magnitude (Physique 2b). Second, the contact area was considerably reduced in size, with only a few dynamic contact points visible, likely reflecting the tips of microvilli contacting the SLB. In this case, no clear increase in the contact area was observable; while some cells responded by the formation of a lamellipodium, others did not show any Mirin detectable alteration of the contact phenotype (Physique S2). We then decided the pressure exerted by T-cells pressure onto the cantilever. According to Newtons third axiom (i.e., actio est reactio) , such pressure directly corresponds to the total normal pressure exerted via a T-cell onto the SLB in the opposite direction. When confronting T-cells with Mirin highly stimulatory SLBs (high levels of I-Ek/MCC as well as ICAM-1 and B7-1), we observed predominantly pulling forces with a magnitude in the single-digit nN range (Physique 4, first row; see Physique S4 for exemplary traces). The onset of pressure exertion.