CONJUSOME ISOLATION

 

Day 1:

1.      In a laminar flow hood, add several drops of cell culture from two different mating types (Cnj I and V work best) into separate flasks containing 25 mL of sterilized NEFF media.

2.      Add 25 µL of 1000x paromomycin to each flask as well. Ideally, the final culture density should be 600k-700k cells/mL on day 3, so the number of drops to add varies on the cell density of the stock culture.

3.      Incubate the flask cultures overnight at 32º C.

 

Day 2:

1.      Make two 1 L flasks of NEFF. Cover the flasks tightly with aluminum foil and autoclave tape, and autoclave for 30 minutes.

2.      Use the Coulter Counter to determine the cell densities of your flask cultures. After turning the counter on (and flushing the aperture and turning the machine off and on again, if necessary), add 1 mL of culture to 19 mL of fixative solution, then put the sample tube into the recess on the platform and raise the platform so the sensor is completely immersed.  Hit “Start” and the machine will count.  Count three times, average these numbers, and multiply by 20 to obtain cells/mL.

3.      Enough culture should be added to each 1 L flask to allow them to reach about 700k cells/mL sometime during the following day. Assume that the cell density of each flask double every three hours.

4.      Add 1 mL of 1000x Pen-Strep to each 1 L flask before incubating at 32º C.

 

Day 3:

1.      Once the flask cultures have reached the appropriate density, wash the cultures with Tris media and incubate them overnight in 100 mL Nunc plates. If the cell counts are low, pellets can be concentrated while they are being washed.

2.      After determining the cell densities of each flask, add 50 mL of the lower density culture to several 50 mL centrifuge tubes, and spin them for two minutes at setting 5. 

3.      Pour off the NEFF media, loosen the pellets, and refill the tubes with 50 mL of cell culture (only refill with more culture if the cell densities were low).

4.      Spin the cells again, pour off the NEFF media, loosen the pellet, and resuspend the cells in 50 mL of sterile Tris media.

5.      Spin the cells again, pour off the Tris, and resuspend in fresh Tris.

6.      Pour the contents of two centrifuge tubes into a 100 mL Nunc plate.

7.      Continue to wash cells in Tris (and concentrate them if need be) until the contents of the flask are exhausted.

The procedure is the same for the higher density culture, except less culture is

added to the centrifuge tubes during each spin; the amount used is (lower cell den

sity/higher cell density * 50) mL. Fill enough Nunc plates with culture from the

high density flask to equal the volume from the low density flask. Incubate the

plates overnight at 32º C.

            Make two sucrose solutions, 55% (55 g/100 mL) and 70% (70 g/100 mL) in

deionized water. Keep cool overnight.

 

Day 4:

1.      Put the SW28 centrifuge rotor and associated centrifuge tubes and holders in the cold room.

2.      Use the Coulter Counter to determine the cell densities of the Nunc plates of different mating types.

3.      To each Nunc plate with a lower cell density, add (lower concentration/higher concentration * 100) mL to a higher cell density culture of a different mating type.

4.      Mix the mating cultures and incubate them at 32º C.  Examine pairing after 2 hrs.  Abort experiment if pairing is low.

5.      6.5 hours after mixing, add CdCl₂ solution up to 0.2 µg/mL to induce fluorescence.

6.      Fill 6 ultracentrifuge tubes with 15 mL of 70% sucrose. On top of this, gently layer 15 mL of 55% sucrose. The tip of the serological pipette should barely break the meniscus of the solution as lighter sucrose is added. Ensure that the interface of each sucrose gradient is clean, with no visible whorls.

7.      8.5 hours after mixing, if fluorescence is good, pellet the cells from each mating culture in the IEC clinical centrifuge for 2 min. on setting 5, resuspending them in an equal volume of cold 2x lysis buffer. Consolidate all pairs into onej 15 mL centrifuge tube (final vol about 2 mL). (note: dibucaine treatment option).

8.      As quickly as possible, sonicate with the Soniprep 150 for 5 seconds at 13 microns and 23 KHz, consolidate five tubes into one, and spin in the IEC clinical centrifuge on #7 for 15 minutes.

9.      Continue the consolidation as long as necessary.

10.  Add equal volumes of lysate to opposite centrifuge tubes and balance their weights to within one hundredth of a gram of each other. Spin overnight (12-16 hours) at 28k rpm and 4º C.

 

Day 5:

1.      Using cannula, suck up the cellular goop at the interface, taking care to suck up as little empty sucrose as possible.  Examine a drop on fluorescence microscope.  If no GFP-conjusomes visible, abort.

2.      Consolidate the enriched conjusomes and some cold lysis buffer in an ultracentrifuge tube and vortex the contents. Spin at 20,000 rpm for one hour.

3.      Pour off the supernatant until there is about 1 mL of fluid left in the tube.

4.      Resuspend the pellet by vortexing and sonicating.

5.      Store the conjusomes by flash freezing them in liquid nitrogen in a 1 mL cryotube and store in a freezer.

2x Lysis Buffer recipe (makes 1 L):

 

0.3 M       KCl                  22.37 g

2 mM       EGTA              0.7608 g

2 mM       EDTA              0.7440 g

100 mM   MgCl₂              20.331 g

0.6 M       Sucrose            171.15 g

50 mM     Tris                  121.14 g

2%           Triton X-100               

 

Inhibitors:

 

2 mM o-phenanthroline (200 mM stock in methanol)

2 mM N-ethylmaleimide (400 mM stock in methanol)

 

To make stock solutions:

o-phenanthroline: 5 g in 138.73 mL methanol

N-ethylmaleimide:             5 g in 99.90 mL methanol

 

note: it’s possible the 5g of N-ethylmaleimide won’t go into the methanol very

easily—it may be necessary to use a 200 mM stock solution rather than 400 mM.