TITLE: Subcellular fractionation of suspension Chinese Hamster Ovary (CHO) cells 
AUTHORS: Saumel Perez Rodriguez, María  De Jesús Ramírez-Lira, Tune  Wulff, Bjørn  Gunnar Voldbor, Octavio  T Ramírez, Mauricio  A Trujillo-Roldán, Norma  A Valdez-Cruz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Subcellular fractionation is a widely adopted technique to study cellular biology, and localization and functions of several proteins </div><div class = "text-block">⁠</div><div class = "text-block">, and, more recently, it has been applied previous to proteomics to quantify low concentration proteins, and characterize many subcellular organelles </div><div class = "text-block">⁠</div><div class = "text-block">. Since CHO cells play a central role for recombinant protein (RP) production, a deeper study of their cellular biology is mandatory in order to construct new cell sub-lines with a more robust RP expression phenotype. Therefore, the goal of the present protocol is to provide the necessary basis for separation of subcellular organelles from suspension Chinese hamster ovary (CHO) cells for a subsequent proteomic study of isolated fractions. Differential and isopycnic centrifugation based sections are presented for enrichment of different subcellular compartments, together with a precipitation section for recovering of proteins from isolated fractions. Isopycnic centrifugation is based on discontinuos sucrose gradients, of which one is adapted from the literature for the separation of nuclear and mitochondrial precipitates </div><div class = "text-block">, and the other is designed in our laboratory for the separation of the microsomal precipitate. This protocol provides a novel discontinuous sucrose gradient for the separation of the components of the microsomes, which increases the separation efficiency with respect to previously reported protocols </div><div class = "text-block">⁠. </div></div>

[STEPS]
?. [Cell disruption]
Quantify cell concentration and viability during cell culture to reach 300-700 millions of viable cells, with a viability higher than 90%.
?. [Cell disruption]
Centrifuge cells at , in  conical tubes.
Centrifuge: 185 34, 5 min, 4 10
50 mL
?. [Cell disruption]
Discard supernatant gently to avoid any cell lost.
?. [Cell disruption]
Resuspend cellular pellet to about 3-7 x 107 cells/ml in  of cold phosphate buffer (NaCl, KCl,  Na2HPO4,  KH2PO4).
10 mL
Calculate total viable cells present in the pellet by multiplying collected culture volume and viable cell concentration. Pool as many cell pellets as necessary to complete the total number of viable cells required for the procedure.
?. [Cell disruption]
Centrifuge cells at , in conical tubes.
Centrifuge: 185 34, 5 min, 4 10
50 mL
?. [Cell disruption]
Discard supernatant gently.
?. [Cell disruption]
Resuspend cell pellet in  of cold phosphate buffer.
10 mL
?. [Cell disruption]
Repeat steps 5 and 6 once.
?. [Cell disruption]
Resuspend cell pellet in  of hypotonic buffer (  HEPES,   EDTA, ).
8 mL
?. [Cell disruption]
Incubate the cell suspension during , inverting tube every .
on ice
?. [Cell disruption]
Add PMSF to a final concentration of  and of prepared Protease Inhibitor Tablets to every 350 millions of cells.
500 µl
To prepare Protease Inhibitor Tablets, dissolve one tablet in   of sterile MilliQ water, filter through a 0.2 µm filter and distribute    aliquots in 1-2 ml eppendorf tubes. Keep at  until use.
?. [Cell disruption]
Transfer the cells to a Dounce homogenizer, and disrupt them by 25 strokes, .
on ice
Since cell disruption must be optimized for each cell line in order to reach maximum efficiency and organelle integrity, number of strokes should be carefully evaluated in a case by case basis.
Another cell disruption method, such as nitrogen cavitation , sonication  and Balch homogenizer ⁠, can be evaluated, depending on availability.
?. [Cell disruption]
Restore isotonicity immediately by adding  of 45% (w/v) sucrose solution, prepared in the hypotonic buffer, to the cell homogenate.
2 mL
?. [Cell disruption]
Transfer disrupted cells back again to  tubes and homogenize this suspension by inversion.
50 mL
?. [Differential centrifugation]
Distribute the cell homogenate in  aliquots into  microcentrifuge tubes.
1 mL
1.5 mL
?. [Differential centrifugation]
Centrifuge at.
Centrifuge: 3000 34, 10 min, 4 10
?. [Differential centrifugation]
Collect supernatant and distribute it to new  microcentrifuge tubes. Tag the pellet as Nuclear Fraction and maintain it .
1.5 mL
on ice
?. [Differential centrifugation]
Centrifuge the collected supernatant at .
Centrifuge: 9000 34, 10 min, 4 10
?. [Differential centrifugation]
Collect and pool the new supernatant in , Open-Top Thinwall Ultra-Clear/Polypropylene Tube, 14 x 89 mm. Tag the pellet as Mitochondrial Fraction and maintain it.
13.2 mL
on ice
?. [Differential centrifugation]
Centrifuge the pooled supernatant at , in a SW40Ti rotor, using an Optima XE-90 Ultracentrifuge.
Centrifuge: 100000 34, 60 min, 4 10
Any other ultracentrifuge compatible with this rotor and running settings can be used.
Read all product manuals and consult with Beckman Coulter-trained personnel before attempting to operate the instrument. Do not attempt to perform any procedure before carefully reading all instructions. Always follow product labeling and manufacturer’s recommendations. If in doubt as to how to proceed in any situation, contact your Beckman Coulter Representative.
?. [Differential centrifugation]
Collect the supernatant in  conical tubes. Tag the pellet as Microsomal Fraction, and supernatant as Cytosol, and maintain both .
15 mL
on ice
?. [Differential centrifugation]
Precipitate proteins from supernatant (cytosol) immediately, as detailed below, and storage protein pellet at until use.
-80 °C
If the proteomic analysis will make use of the differential fractionation samples, without any further separations, storage nuclear, mitochondrial and microsomal pellets at  until use. In case that these pellets will be separated in density gradients, do not freeze these samples, and proceed to separation immediately after their obtaining.
?. [Isopycnic centrifugation]
Prepare discontinuous sucrose gradients  by depositing the indicated volumes on the tube wall, drop by drop, slowly.
on ice
Use a gradient of   of 60% (w/v) and   of each solution of 55, 40 and 30% (w/v) sucrose, placed from the bottom to the top of the tube, for separation of nuclear and mitochondrial pellets ⁠. A second one formed by   60% (w/v) and   of each solution of 45, 35, 30 and 10% (w/v) sucrose, placed from the bottom to the top of the tube, will be used for separation of microsomal pellet. Sucrose solutions should always be deposited in decreasing order of concentration. The interface between these solutions should always be visible, except in the case of 30/35% where it is difficult to distinguish. Discard gradients if interfaces are not clearly visible. Use  , Open-Top Thinwall Ultra-Clear/Polypropylene Tube, 14 x 89 mm to prepare gradients. All solutions should be kept on ice throughout the procedure, and prepared according to the percentage of sucrose indicated in each case (w/v), in a buffer with a final composition of   HEPES and   EDTA,  .
?. [Isopycnic centrifugation]
Pool all the mitochondrial pellets by resuspending them gently in  of isotonic buffer [ sucrose (w/v),  HEPES,  EDTA, ].
1 mL
?. [Isopycnic centrifugation]
Divide nuclear pellets in 4 groups, and pool each group by resuspending them gently in  of isotonic buffer.
1 mL
?. [Isopycnic centrifugation]
Resuspend the microsomal pellet in  of isotonic buffer.
0.5 mL
?. [Isopycnic centrifugation]
Add each resuspended pellet to the top of the corresponding sucrose gradient and centrifuge at  (max), in a SW40Ti rotor, using an Optima XE-90 Ultracentrifuge, with maximal and medium acceleration and deceleration rates, respectively.
Centrifuge: 217874 34, 180 min, 4 10
Any other ultracentrifuge compatible with this rotor and running settings can be used.
?. [Isopycnic centrifugation]
Collect visible bands in each gradient by using a Pasteur pipette,  micropipette tips, a peristaltic pump, or by tube puncture or any other method suitable for fraction collection.
1 mL
Three bands should be seen on each gradient, except for the nuclear gradient, where 4 bands are resolved.
The densest band on the nuclear gradient, located at the bottom of the tube, should be discarded as it represents cell debris.
?. [Isopycnic centrifugation]
Precipitate proteins from each fraction collected from sucrose gradients by the protocol detailed below, and storage protein pellets at  until use.
-80 °C
?. [Protein precipitation]
Add  of  NaCl to  of sample in  conical tubes, and homogenize by vortex.
260 µl
10 mL
50 mL
This section has been optimized to precipitate complex protein mixtures from CHO cells ⁠. The obtained pellet can be shipped to a Proteomic Facility, at , for its processing and analysis. Total volumes should be changed according to available sample, always maintaining the indicated proportion of solutions.
?. [Protein precipitation]
Add  of cold 80% (v/v) acetone and homogenize by vortex or inversions. Acetone should be kept at , at least  before precipitation.
40 mL
-20 °C
Acetone percentage can be increased to 100% (v/v) if protein recovery is not enough for proteomic analysis.
Use gloves, safety glasses and protective clothing. Do not eat, drink or smoke during use. Keep away from heat, hot surfaces, open flames, sparks. Keep container tightly closed.
?. [Protein precipitation]
Incubate at during .
-20 °C
Incubation for a longer time could be necessary if protein recovery is not enough for proteomic analysis.
?. [Protein precipitation]
Centrifuge at . Discard supernatant.
Centrifuge: 8228 34, 30 min, 4 10
?. [Protein precipitation]
Add  of 80% (v/v) acetone and homogenize by vortex or inversions.
40 mL
?. [Protein precipitation]
Centrifuge at . Discard supernatant.
Centrifuge: 8228 34, 30 min, 4 10
?. [Protein precipitation]
Repeat steps 34 and 35.
?. [Protein precipitation]
Centrifuge at  and discard all supernatant carefully by using a micropipette.
Centrifuge: 8228 34, 1 min, 4 10
?. [Protein precipitation]
Dry the pellet at  during .
-20 Room temperature
Do not over-dry the precipitate as this decreases the solubilization of the proteins in a later step.
?. [Protein precipitation]
Tag and seal the tube to be shipped to Proteomic Facility.