The overall goal of this procedure is to visualize structures such as F actin in the NK cell lytic synapse using TED microscopy. This is accomplished by first recapitulating the synapse on glass using activating antibody. The second step of the procedure is to fix perme and stain the cells for structures of interest.
Next, the slides are imaged by laser scanning confocal microscopy, followed by application of stead depletion for increased image resolution. The final step is to process the images using a deconvolution algorithm. Ultimately, results can show sub 100 nanometer resolution of cellular structures thanks to dual color stead and endoscopy.
This method can help answer key questions in the cell biology field, such as a relationship between the cytoskeleton and exocytosis. In our case, we are interested in the secretion of specialized lysosomes termed lytic granules by natural killer cells. Generally, individuals new to this method will struggle because there are multiple variables that can be adjusted in the application of the TED depletion laser.
An inappropriate application of the depletion beam will result in a worsening of instead of improvement in resolution In preparation, warm 30 milliliters of RPMI media with 10%FCS and separately. One milliliter of BD CY effects cyop perm both to 37 degrees Celsius. Then begin the process of coating the cover slips with antibodies.
Take number 1.5 cover slips and using a PAP pen. Make dime-sized squares on the cover slips. Prepare one square for each tested condition.
Load each circle with 200 microliters of antibody at five micrograms per milliliter in PBS. For NK 92 cell activation, use anti CD 18 and anti NK P 30 antibodies. Incubate the loaded cover slips at 37 degrees Celsius for half an hour, and then wash them off with a dip in room temperature PBS proceed immediately with adding the cells for each tested condition, 500, 000 cells must be ready to use.
Wash the cells in 10 milliliters of the prewarm media and then resuspend them in the same warmed media. At 2.5 million cells per milliliter with the cells fully in suspension, decant 200 microliters into each antibody square on the cover Slips incubate the cover slips with cells at 37 degrees Celsius with 5%CO2 in this case. 20 minutes is enough time to polarize the granules in the NK cells after the incubation wash, the cover slips gently in PBS at room temperature.
Begin with adding a microliter of TRITTON X 100 to a milliliter of warm BD CY effects cyto perm solution. Then vortex. Apply 200 microliters of this solution to each condition square on the cover slips.
Then place the cells in the dark at room temperature and wait 10 minutes. When the incubation is finished, gently wash the cover slips in staining buffer. Then dry the edges of the surrounded regions with a cotton swab.
Next, add 200 microliters of the primary antibody in staining buffer to the surrounded regions, and let the cover slips incubate in the dark for half an hour. Some good secondary antibodies for stead imaging include Alexa floor 4 88 Pacific orange and Horizon V 500. Each of these work well at a one to 200 dilution.
After the incubation wash, the cover slips in staining, buffer, and dab them dry. As before, apply 200 microliters of the secondary antibody to each condition square. Then let them sit in the dark for another 30 minutes.
At this point, it's possible to add additional antibodies after additional washes. For example, Phin can be applied at one to 200 to detect f actin. For a mounting medium, choose prolong over vector shield, which is not compatible with stead.
If phin staining is used, avoid using two two thio ethanol while however is okay, now apply 10 to 20 microliters of mounting medium to a slide. With the cover slip inverted, gently lower it into the medium, avoiding air bubbles. Then incubate the slide for 24 hours.
With the cover, slip up the next day, seal the cover slip with nail polish and proceed with imaging. Start up the lasers and software. The stead depletion laser should warm up at 100%power and be aligned.
Using the eyepiece focus on the sample, then turn to the software. Scan the first channel, the one with the longest wavelength. Floor four, optimize the laser's power, the excitation beam position, and the detector range.
Avoid setting above 100. Next, capture a confocal image and check for pixel. Saturation, line, and frame averaging will both enhance pixel resolution.
The goal is to be below 30 nanometers per pixel. Some saturation is acceptable for stead. Next, apply the stead depletion beam at 50%power and capture an image.
If the resolution improves, more power can be applied. Further, adjusting the excitation laser power or the line average or the gain could also improve the resolution. To reduce the background apply 0.3 nanoseconds of time gating and adjust upward.
The new resolution should be estimated using a full half width maximum calculation. Once satisfied, repeat these steps with the second channel when all the channels are tweaked. Check for spectral overlap by imaging single stain controls with all the scan sequences.
Mild overlap can be corrected with spectral unmixing by the software but is best avoided or kept to a minimum. Now, acquire images for quantitative imaging. Obtain at least 20 images per condition according to the experimental conditions.
To devolve the saved images, open the files in the appropriate software. Check the parameters of each channel, specifically the excitation and admission spectra, the stead depletion emission, and the imaging direction. Then apply the software's deconvolution calculations.
Default settings usually suffice, but the signal to noise will vary between floor force, so that parameter needs to be set manually for the experiment. There are a few common pitfalls that may lead to suboptimal resolution with stead. One is under sampling.
This leads to graininess and a loss of pixel information such as in these f actin filaments. Increasing the line or frame averaging often remedies this issue. Another issue is bleaching or oversampling caused by lengthy pixel.
Dwell time usually due to excessive line averaging over scanning prior to image acquisition may also be the culprit and can be corrected using a smaller scan before acquiring images or by using a higher scan speed. Reducing the laser power can also help with everything working. The image will improve dramatically.
Deconvolution will even further improve the image. Both quantitatively and qualitatively. Sub 100 nanometer resolution should be routinely obtained While attempting this procedure.
It's important to remember to perform stead beam alignment prior to each experiment and approximately once an hour thereafter following this procedure. Other methods like quantitative analysis can be performed in order to answer additional questions regarding colocalization, subcellular localization and molecular interactions.
