I work with mouse OT-I CD8+ T cells which have been activated in vitro with peptide-MHC and B7 proteins for 3 days. I've been trying to standardize protocols to fix these cells using paraformaldehyde (PFA). I usually add equal volume of 2x concentrated PFA to achieve a final concentration of 3.5% PFA to the cells, and fix these at 37C for 10 minutes. The PFA I use is diluted from a 16% PFA stock (aqueous solution from Electron Microscopy Sciences, catalog# 15710) in 1X PBS. After the PFA incubation is complete, I wash the sample thrice with 1X PBS, with 5 minutes between each wash.
When I viewed the cells fixed using the above protocol using Interference Reflection Microscopy, the cells seemed to have "cavities" or "pits" (sample image attached). Does anyone know what causes these "cavities" and how can I modify my protocol to prevent these from occurring? Fixation of OT-I T cells using 4% PFA for 10-15 minutes seems to be used pretty commonly in the literature, so I'm not sure what's going on.
I would really appreciate any suggestions regarding this! Thanks!
Dear Aashli!
Please You look at following article:
Visualizing Dynamic Microvillar Search and Stabilization during Ligand Detection by T cells
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364556/
Cell preparation for imaging, fixation and staining
To prepare OT-I T cells for imaging, live cells were harvested using Ficoll-paque, washed with complete RPMI, and then held at 37 °C.
For LLS fixed cell imaging on activating lipid bilayers, 2×106 OT-I T cells were stained with 2.5 μg of CD45 non-blocking monoclonal antibody conjugated to Alexa Fluor 647 on ice for 30 minutes and then rinsed once with complete RPMI. 5×105 cells were added to a 5 mm in diameter round coverslip sitting in an imaging well of 8-well Nunc Lab-Tek II chamered coverglass, allowed to bind for 6 minutes, and then fixed with 20 mM HEPES, 0.2 M Sucrose, 4% paraformaldehyde (PFA) and 0.01% Glutaraldehyde for 10 minutes. The fixed cells were washed with 8 mL of PBS for imaging.
Synaptic Contact Mapping (SCM) and Interference Reflection Microscopy (IRM)
The TIRF microscope is based on a Zeiss Axiovert 200M with a manual Laser TIRF I slider (19). To image nanocontacts and TCRs, image sequences consisting of TCRs (TIRF), QD-streptavidin (widefield), and interference reflection microscopy (IRM, widefield) images were collected. For TIRF images, a 488 nm Obis laser (Coherent) was used for excitation of Alexa Fluor 488-labeled TCRs (or CD3ζ-GFP or ZAP70-GFP), and a 561 nm Calypso laser (Cobolt) was used for Alexa Fluor 568-labeled TCRs.
SCM used a combination of TIRF detection of TCR or GFP fusions together with wide-field excitation/emission of Quantum dots. Widefield QD images were acquired using a 405/10x excitation filter (Chroma Technology) in a DG-4 Xenon light source (Sutter). TCR and QD fluorescence were split using a DV2 with a 565 nm long-pass dichroic and 520/35m and 605/70m emission filters (Photometrics). Split images were collected using an Evolve an electron-multiplying charged-coupled device (emCCD) in quantitative mode (Photometrics). IRM images were acquired using a 635/20x excitation filter (Chroma Technology), and reflected light was collected onto the long-pass side of the DV2 imaging path. For time lapse image series, image sequences were typically acquired at 1 second intervals.
Pay attention to the protocol of fixation with pharmacaldehyde and especially to the settings, filters of interferometric microscopy Most likely, your pits and cavities are the result of microscope settings, since the principle of the method Light is reflected from each boundary where the refractive index changes, for example, from the boundary between the glass of the dish and the solution poured into it, as well as from the boundary between the cell and the culture environment. Thus, light incident at a right angle or close to a right angle on the glass - water interface, and then on the water - plasmalemma interface, gives two reflected waves that differ in phase. These two reflected waves, since they have different phases, will give an interference pattern.
Figure 6.
Cytoskeletal independence of TCR-mediated protrusion stabilization.
(A) Bilayer-bound QD605-streptavidin, TCR and IRM time lapse images of an OT-I IS during Latrunculin B (LatB) challenge. 1 μM LatB was added after acquisition of the t = 0 sec image. Scale bar: 5 microns.
I would recommend storing the stock solution at -20 and ensure minimal contact with light. That said, I am not sure this is entirely artifact that you are observing. As per your protocol, these cells have been activated on siinfekl. I would expect granules containing perforin and granzyme at this time point.