Pulse-chase labeling of the cell wall in mycobacteria
We use this protocol to pulse chase label the cell wall in mycobacteria. This experiment allows us to establish the sites of new growth in the chase portion of the experiment. For examples, please see above and Aldridge et al. Science 2012.
Make up and aliquot the dye
(Alexa Fluor® 488 Carboxylic Acid, Succinimidyl Ester, mixed isomers (1 mg)
1.Add 250 uL of anhydrous DMSO to the 1 mg tube of dye. (air and water are not friendly to the dye)
2.Divide the solution into 5-10 uL aliquots, double bag the tubes, and store in -80C. These will store for a few months, but the reaction suffers the longer they are stored. Do not freeze thaw.
Prepare your cells
1.Staring with a log phase 10 mL culture, pellet and resuspend in 10mL PBS+0.2% Tween or TBST. Spins are done at 2000 RPM for 4 minutes.
2.Pull as much of the supernatant off as possible to remove sugars and proteins from the media. Resuspend in 500uL of TBST or PBST.
3.Add a 5 uL aliquot of dye, flick to mix and spin immediately. Do not let the dye sit or incubate on the cells. Remove the supernatant.
4.Wash with TBST, PBST, or media.
5.Resuspend in normal growth media for the chase portion of the experiment. We either fix part of the culture with 4% PFA at multiple time points or perform live cell imaging to observe the growth pattern
Microfluidics for live cell imaging of mycobacteria
In collaboration with Mehmet Toner, Daniel Irimia, and Marta Fernandez-Suarez at Massachusetts General Hospital, we have designed microfluidic devices that enable us to culture and image mycobacteria for extended periods of time. Our devices meet the following essential design criteria:
1.Homogeneous growth environment: a constant infusion of media feed cells in the growth channels through diffusion.
2.Nonfunctionalized growth surface: functionalizing cover glass to keep cells in the focal plane with poly-l-lysine, for example, is known to disrupt the growth characteristics of bacteria. See Colville et al. Langmuir 2010 (http://pubs.acs.org/doi/abs/10.1021/la902826n) and below for more information about the effects of lysine. Our device allows us to physically constrain the cells to the focal plane with a short depth of the growth channels.
Time lapse imaging of mycobacteria grown in a microfluidic device on cover glass functionalized with poly-l-lysine. The old cell wall material was pulse labeled (green) and new growth is observed in the red pseudo-colored brightfield image. Poly-l-lysine coating causes cells to stick to the cover glass, preventing sister cells from v-snapping and moving away from each other at division. Lysine coating also alters the growth pattern of mycobacteria, which exhibit asymmetric polar growth on uncoated glass (Aldridge et al., Science, 2012). These time lapse images show examples of symmetric, bipolar growth of mycobacteria grown on lysine-coated glass (blue and yellow arrows mark the old and new poles of one cell).
3.Fluorescent tags are not necessary: the short depth of the channels allows us to visualize the cells in a single plane by microscopy; this enables us to image the cells without necessitating the use of protein-fluorescent protein fusions that may alter protein function and cell behavior. See Landgraf et al. Nature Methods 2012 for more information about mislocalization of fluorescent fusion proteins.
We are happy to provide sample devices to academic labs. Please contact us for more information.