Reagents:

Four to six, 10-12-week-old male FVB/NJ mice: JAX stock number 001800.

Cell strainers: pluriSelect pluriStrainer®: 100 µm (SKU 43-50100-51), 70 µm (SKU 43-50070-51), 40 µm (SKU 43-50040-51) and 30 µm (SKU 43-50030-03)

1X RPMI-1640 Medium: Sigma R0883-6X500ml.

Antibiotic and Anti-mycotic: 100X Anti-Anti (ThermoFisher 15240062 Antibiotic- Antimycotic (100X)).

Fetal bovine serum (FBS): ThermoFisher 10082-147.

KREBs Medium: -1 bottle Krebs buffer powder (Sigma Aldrich Product Number K3753)

-20mL 50X Amino Acid Solution (Gibco 1130-051)

-10mL 100X NEAA (Gibco 11140-050)

-10mL 100X glutaMAX (Gibco 35050-061)

-CaCl₂ (0.14g)

-NaHCO₃ (2.1g)

-0.14% BSA (14g)

- ~800mL ddH20 (fill to 1000mL after pH w/ 1M NaOH to pH 8.0)

-25X collagenase: dissolve 25mg of Collagenase in 4mLs RPMI or Krebs (Collagenase from Clostridium histolyticum Sigma-C6885-100MG), syringe filter and store 1mL aliquots at -20°C

-50X DNase: mix 5mLs glycerol +5mLs RPMI +25mg DNAseI (DNAse I lyophilized C6885-100MG) syringe filter and store 1 mL aliquots at -20°C

- Trypsin-EDTA (0.25%): StemCell - Enzymatic cell dissociation reagent Catalog #07901

Dissociation Medium: Make a 2X dissociation medium by mixing 2 mLs of 25X collagenase, 1 mL of 50X DNAse and 22mL of RPMI. Remember that when adding the solution to the chopped tissue you have to adjust the volume to make the final solution be 1X. For example in step 8 bellow, if you have 3 mL of cut tissues you will add 2 mL of RPMI and 5mL of 2X dissociation medium.

Trypsin DNAse Medium: Add 2mLs of 50X DNAse to 50mLs of Trypsin-EDTA (0.25%).

Complete RPMI Medium: RPMI-1640 supplemented with 10% FBS and Antibiotic and Anti-mycotic.

Procedure:

Fill water bath and set water bath and incubators to 35C, warm media and set centrifuge to RT

Euthanize mice, remove the epididymis to 10cm petri-dished containing prewarmed (35°C) with KREBS media. Dissection does not have to be perfect – just get all samples into media
Once all the epididymides have been collected, remove any major fat, blood vessels, and trim the organ capsule and any attached tissue not from the epididymis tubules moving clean organs to a new 10 cm plate containing 10 mL of Krebs media pre-warmed to 35°C.
Separate caput, corpus, cauda, and vas and place each section on new dishes containing warm media. Make few small incisions to allow sperm to escape. If possible, leave dishes shaking while performing the procedure for the different epididymides. At the end of this step you should have 4 dishes, each containing one type of segment; one plate with all the caputs, one with all the corpus, one with all the cauda and one with all the vas deferens.
Transfer the segments to another 5 cm petri dishes with about 2 mL of Krebs media. Using curved scissors cut each group of segments into small pieces - roughly 1- 5 mm pieces - and transferred all the media and tissue to a 50 mL conical tube.
Add KREBS media to a final volume of 30 mL and allow samples to decant while in an upright position at a 35°C incubator for three to five minutes and discard supernatant.
Add fresh media (~30mL) and resuspend gently using wide bore transfer pipettors. Allow samples to decant while in an upright position at a 35°C incubator for three to five minutes and discard supernatant.
These steps were repeated until supernatant looked clear, with the last wash being of RPMI.
Once dissected samples were cleared of any visible sperm, and the final RPMI wash performed, there was about 3 mL of the RPMI sample mixture. The tissue and left over RPMI were transferred to small 25 mL glass Erlenmeyer flask containing 5 mL of 2X dissociation media and conical tube was further rinsed with 2mL RPMI.
The 4 Erlenmeyer flasks containing 10 mL of sample in dissociation media were placed in a 35°C water bath with rotations of 200 rpm for 30 to 45 minutes. Afterwards, samples were transferred to a conical tube and allowed to settle for ~5 minutes. Supernatant was discarded, leaving four to five mL of sample solution to be returned to the Erlenmeyer flask.
Add 10 mL of trypsin DNAse to each Erlenmeyer flask and return them to the 35°C water bath with rotations of 200 rpm for 35 minutes.
After 35 minutes the samples were pipetted up and down until there were no observable pieces of tissue and allowed to incubate in the water bath for an additional 15 to 20 minutes.
Once cell disaggregation was achieved add 2 mL of FBS to inactivate the trypsin.
Transfer samples to new falcon tube while passing the cell solution through a series of 100 mm, 70 mm, and 40 mm cell strainers, and adding Complete RPMI media to a final volume of 30 mL.
Centrifuge samples for 15 minutes at 400 rcf (g) at room temperature (RT). Discarded supernatant and add up to 25 mL of Complete RPMI media and spin cells down for 5 minutes at 800 rcf (g) at RT centrifugation step. Discard supernatant
Add up to 2 mL of PBS, and transfer cells to a 2 mL microfuge tube.
Cells were spun down by a 900rcf (g) for 3 minutes and supernatant discarded. Pelleted cells were washed once more with up to 2mL PBS followed by similar spin and supernatant discarded.
Cells were resuspended in 600 mL of PBS and placed on ice.
Two measurements of viability using 10mL of cells plus a cell viability marker (tryphan blue), was used to determine cell viability and concentration using an automated cell counter (Bio-RAD TC20TM ).

Cells were maintained on ice and samples deemed good if we obtained a (>80% survival)

Obs: The Rando lab has switched to using only IMDM (IMDM GlutaMAX, +25mM HEPES, +3.024g/L Sodium Bicarbonate (Gibco Cat# 31980030)) media in all steps instead of KREBS and RPMI and added the following enzymes to the dissociation media to reduce incubation time (0.75 mL of 10x Collagenase/Hyaluronidase (StemCell Cat# 0791) and 0.5mL of 100X solution of Collagenase/Dispase (Sigma Cat#11097113001)) and has been obtaining similar results.