The workflow consists of growing cells in strictly controlled environmental conditions, rapidly chilling them using melting water/ice and fixing with pre-optimized concentration of formaldehyde and incubation time. The fixation reaction is then quenched using excess of primary amino group-containing compounds, such as glycine or tryps. Cells are collected, washed and can be stored frozen.
Fixed cells can be mechanically or chemically lysed, and the lysate is used for separation of the ribosomal fractions based on their molecular size, sedimentation properties or affinity markers. The approach results in versatile material usable for immuno assisted purification and detection, enrichment of complexes containing specific factors in electron microscopy. Upon crosslinked deep blocking, RNA amplification or hybridization based analyses, RNA sequencing and mass spectrometry become possible.
Set up a one-liter yeast culture in an orbital shaker with a starting optical density no more than 0.05 absorbance units at 600 nanometers and use peptone and dextrose media at 30 degrees celsius. Set up a preparative centrifuge with compatible rotor and centrifuge bottles for pelleting the liquid suspension of yeast cells at 5000 G at four degrees celsius. Keep record of the optical density of the growing cells.
It must be between 0.6 to 0.8 absorbance units and 600 nanometers at the time of collection if the exponential growth phase is of an interest. Once the cells are ready, set up an icebox inside the chemical fume hood with a baker containing 250 grams of clean crushed water ice and show you also 25 milliliters trypsin and freshly purchased methanol stabilized 37%formaldehyde solution inside the hood. Pour the one latest cell culture into the baker containing the ice.
Then add 75 milliliters of 37%formaldehyde to a final concentration of 2.2%weight over volume intensely stir the mixture until the ice melts. Once the ice is melted, set up a timer for 10 minutes After incubating for 10 minutes, transfer the culture into the pre-cooled centrifuge bottles and pellet the cells by centrifugation at four degrees celsius, 5000 G for five minutes. Whilst the spin is, on pre-cool a 50 milliliter tube and keep freshly prepared buffer A containing glycine to neutralize any remaining formaldehyde on ice.
After centrifuging, place the centrifuge tubes onto ice with the pellet side in contact with the ice. Bring the tubes into the fume hood and discard the supernatant into a formaldehyde waste container. Re-suspend the cell pellet from old tubes in 20 milliliter of buffer A using a 25 milliliter stripette and transferring into a 50 milliliter tube.
Make up the volume to 40 milliliter with buffer A and collect the wash cells by centrifugation of four degrees celsius, 5000 G for five minutes. Discard the supernatant and re-suspend the cell pellet in 40 milliliter buffer A one, which is buffer A not containing glycine to remove glycine contamination. Pellet the cells again by centrifugation at four degrees celsius, 5000 G five minutes.
Repeat the washes with buffer A one, one more time. Discard the supernatant and place the cell pellet on ice. Weigh the tube with the pellet, the wet cell mass should be approximately one gram per one liter of the cell culture.
Fill up polystyrene from box lined with aluminum foil with liquid nitrogen to a depth of approximately three centimeters. Place at 50 milliliter tube upright in the box. Re-suspend the pellet in 550 microliters of buffer A two by pipetting and vortexing for 10 seconds.
Add 10 microliters of 40 units per microliter RNase inhibitor and vortex again for 10 seconds. Using a one milliliter pipette, drip the cell suspension into the 50 milliliter two containing the liquid nitrogen. To prepare for the next step, pre-cool 1.5 milliliter nucleus free tubes on dry ice and 10 milliliters stainless steel grinding jars.
Transfer the frozen cell suspension droplets into the jars using a clean sterile spatula. Submerge the grinding jars into liquid nitrogen for one minute, ensuring the liquid phase remains above the junction. Set up a Cryo mixing mill at 27 Hertz for agitation for one minute.
Agitate the sealed grinding jars at 27 Hertz for one minute in the mixer mill. Re-cool the jars in liquid nitrogen as before and shake for one minute further. Transfer the jars to the ice box containing dry ice along with the 1.5 milliliter nucleus free tubes.
Using a small steel spatula transfer the results in padded grind date into the tubes. Then store the tubes at minus 80 degrees celsius. In two T175 Flasks grow HEK 293 cells to 60 to 70%confluence and Dalbeko's modified Eagle medium and 10%fetal bovine serum at 37 degrees and 5%carbon dioxide.
At least three hours prior to the desired fixation time, replace the media of the T175 flask with precisely 30 milliliters of pre warmed complete media. Prepare an icebox to the brim with crushed water ice and keep in the fume hood along with required buffers, also an ice. To snap chill the cells, remove the T175 flask from the incubator and formally press it against the ice ensuring maximum surface contact.
Inside a chemical fume hood, tilt the flask onto its side so that the media collects on the side opposite to the cells. Pipette 168 microliters of 37%weighed by volume formaldehyde directly into the pooled media. Giving a final concentration of 0.2%formaldehyde.
Immediately mix. Incubate the flasks on ice for 10 minutes further. Pour off the media into an appropriate waste container through the flask side opposite to the cells.
Using a strippete, pipette in 30 milliliters of Dalbeko's phosphate buffered saline without calcium and magnesium ions and additionally containing 50 millimolar glycine gently on the side opposite to the cells. Mix by rocking the flask, return the flash to horizontal position and incubate for 10 minutes more on ice. Pour off the solution through the flask side opposite to the cell and gently add seven milliliters of standard 0.25%trypsin EDTA solution to detach and re-suspend the cells.
Incubate the flask at room temperature for five, maximally 10 minutes. Locate the flask vertically and using a strippete, collect the detached cells by gently washing any remaining from the flask walls and transfer the suspension into a 50 milliliter tube set on ice. Immediately supplement the collected suspension solution with 20 milliliters of complete media and mix by gently flipping the tube.
Pellet the cells by centrifuging the tube at 100 G for five minutes and four degrees celsius. Cell pellet must be clearly visible. Pour off the media and gently re-suspend the pellet in 10 milliliters of Dalbeko's phosphate buffered saline with no glycine.
Centrifuge the tube again at 100 G for five minutes and at four degrees celsius Pour off the wash buffer and re-suspend the pellet in 800 microliters of ice cold Dalbeko's phosphate buffered saline with no glycine, but containing magnesium and calcium ions. Transfer the re-suspended cells into a new low protein binding 1.5 milliliter micro centrifuge tube. Centrifuge the tube at 100 G for three minutes at four degrees celsius.
Carefully discard the supernatant by using one milliliter pipetter. At this stage, the cell pellet can be frozen at minus 80 degrees celsius or proceed to the cell lysis step. In a biosafety cabinet add 300 microliters of lysis buffer based on non ionic, non denaturing detergent, and seven microliters of arginase inhibitor and mix well by pipetting using one milliliter tip.
Carefully attach a 25 gauge needle to a one to three milliliter syringe and vigorously pipette the mixture using at least seven slow upward intake and fast downward exhaust strokes. Discard the syringe and needle into the sharp's bin and repeat the procedure with a 31 gauge needle and 0.3 milliliter syringe. Discard the syringe and needle into the sharp's bin.
Centrifuge the tube at 12, 000 G for five minutes at four degrees to remove the cell debris. Transfer the supernatant into a new low protein binding 1.5 milliliter micro centrifuge tube. Store both the cell debris and lysate at minus 80 degrees celsius.
Translational complexes are sensitive to the ionic composition of the buffers. Omittance of magnesium chloride and addition of EDTA abrogated high sedimentation properties, and while calcium chloride resulted in better result peaks, the improvement was marginal. We thus selected buffer one.
2.2%weight by volume formaldehyde excellently preserved the polysomes and did not decrease the overall yield compared to 4%weight by volume formaldehyde. In mammalian cells, a much lower concentration of formaldehyde of 0.2%weight by volume was used. As higher concentrations resulted in substantial polyribosomal material loss.
15 millimolar EDTA added to the lysate and all subsequent buffers exhibited distinctively lesser destabilization effect on the polysomal fractions, derived from the fixed cells. And this effect was partially replicated with 50 millimolar EDTA with material from 4%fixed cells, resisting, unfolding better. Glucose depletion elicits one of the most dramatic and rapid translational inhibitory effects on yeast.
Both nerve added and locally cause conditions induced polysome disassembly was slightly, but evidently more polysomes retained in low added glucose. Importantly, polysomal material from the fixed cells demonstrates a high distinction between the starved and non-starved cells indicating the suitability of formaldehyde fixation to preserve differences in dynamic translational processes. In approaches such as tacy paysake, it may be additionally insightful to remove small ribosomal subunits that do not co-settle well with the complete rhizomes for which we employed a two-stage ultracentrifugation, allowing the isolation of SSU, LSU, RS, RNase resistant diazomes, DS, and high order polysomes confirmed by electron microscopy of the fixed fractions.
Compared to the non-fixed cells, material from the fixed cells demonstrated elevated presence of labile eIF4A in the fastest sedimenting ribosomal fractions. Using fixed material from eIF4A tapi strain to capture and enrich eIF4A containing complexes with magnetic IDG beads, we were able to observe selective enrichment of the eIF4A compared to beta-actin in SSU and RS, but not LSU fractions from the second gradient upon RNase one disassembly. Compared to other methods of translational rest, the swiftness of formaldehyde action across cell membranes and the indiscriminate nature of cross-links promises the preservation of the maximum diversity of translation complex intermediates.
Our findings conform the usability of rapid formaldehyde fixation to stabilize highly transient complexes and evidences'usefulness in the scenarios of rapid cellular responses to environmental changes or stress conditions.
