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protocols:step-by-step_cryopreservation_protocol [2023/04/26 16:45]
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| **1.** Prepare RMMB cryoprotective medium (100 mL) in the following order:\\ - Combine 20.0 g D-(+)-raffinose pentahydrate (Sigma R7630 or 83400) and 70 mL dH<sub>2</sub>O in a 250 mL beaker.\\ - Then, place the beaker in an evaporating dish (Pyrex 3140) or large beaker containing hot water (~70 ºC) on a stir plate.\\ - Stir the mixture until the raffinose is completely dissolved.\\ | **1.** Prepare RMMB cryoprotective medium (100 mL) in the following order:\\ - Combine 20.0 g D-(+)-raffinose pentahydrate (Sigma R7630 or 83400) and 70 mL dH<sub>2</sub>O in a 250 mL beaker.\\ - Then, place the beaker in an evaporating dish (Pyrex 3140) or large beaker containing hot water (~70 ºC) on a stir plate.\\ - Stir the mixture until the raffinose is completely dissolved.\\ |
| - Add 2.5 g skim milk (Difco #232100) and stir until it is completely dissolved.\\ - Let the solution cool to room temperature and add 3 mL of 1 M Bicine-NaOH (8.0).\\ - Add 6.67 mL absolute methanol (acetone-free, absolute, Certified ACS Reagent Grade), transfer the mixture to a 100 mL volumetric flask, adjust the final volume to 100 mL with dH<sub>2</sub>O, and mix by inversion three to four times. \\ - Transfer the solution to two 50 mL conical tubes and centrifuge at 15,000g for 20 min at 25 ºC.\\ - Transfer the clear supernatant into a clean beaker and aliquot into 1.5 mL microfuge tubes, 1 mL each, or a different convenient volume for daily use.\\ - Store the RMMB solution frozen at -20 or -80 ºC until use.\\ The resulting RMMB cryoprotective solution will contain 20 % (w/v) D-(+)-**R**affinose pentahydrate, 2.5 % (w/v), Difco skim **M**ilk, 6.67 % (v/v), **M**ethanol, and 30 mM **B**icine-NaOH.\\ | - Add 2.5 g skim milk (Difco #232100) and stir until it is completely dissolved.\\ - Let the solution cool to room temperature and add 3 mL of 1 M Bicine-NaOH (8.0).\\ - Add 6.67 mL absolute methanol (acetone-free, absolute, Certified ACS Reagent Grade), transfer the mixture to a 100 mL volumetric flask, adjust the final volume to 100 mL with dH<sub>2</sub>O, and mix by inversion three to four times. \\ - Transfer the solution to two 50 mL conical tubes and centrifuge at 15,000g for 20 min at 25 ºC.\\ - Transfer the clear supernatant into a clean beaker and aliquot into 1.5 mL microfuge tubes, 1 mL each, or a different convenient volume for daily use.\\ - Store the RMMB solution frozen at -20 or -80 ºC until use.\\ The resulting RMMB cryoprotective solution will contain 20 % (w/v) D-(+)-**R**affinose pentahydrate, 2.5 % (w/v), Difco skim **M**ilk, 6.67 % (v/v), **M**ethanol, and 30 mM **B**icine-NaOH.\\ |
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| **2.** Fill a Styrofoam container or cooler (9 inch or 23 cm min. depth) with powdered dry ice made from liquid CO2 (ZIRC E400_RMMB Sperm Cryopreservation & IVF Protocol https://zebrafish.org/wiki/protocols/cryo ) (see Note((A cooling rate between -10 and -15 ºC per minute was determined to be the optimal range for the materials and solutions used in this protocol. Using powdered dry ice with stacked cryovials in a 15 mL Falcon tube will achieve a cooling rate of 10-15 ºC/min. If possible, avoid changing the materials specified in this protocol. If the specified materials are not available (for example, the 15 mL vials or cryovials with the same wall material and thickness as specified here), you need to determine which freezing rate provides the optimal postthaw fertilization rates with the replacements.You may also have to adjust the thawing protocol to match the new freezing rate. The description for making powdered dry ice is included in the online protocol: https://zebrafish.org/wiki/protocols/cryo.\\ \\ ))).\\ | **2.** Fill a Styrofoam container or cooler (9 inch or 23 cm min. depth) with powdered dry ice made from liquid CO2 (ZIRC E400_RMMB Sperm Cryopreservation & IVF Protocol https://zebrafish.org/wiki/protocols/cryo ) (see Note((A cooling rate between -10 and -15 ºC per minute was determined to be the optimal range for the materials and solutions used in this protocol. Using powdered dry ice with stacked cryovials in a 15 mL Falcon tube will achieve a cooling rate of 10-15 ºC/min. If possible, avoid changing the materials specified in this protocol. If the specified materials are not available (for example, the 15 mL vials or cryovials with the same wall material and thickness as specified here), you need to determine which freezing rate provides the optimal postthaw fertilization rates with the replacements.You may also have to adjust the thawing protocol to match the new freezing rate. The description for making powdered dry ice is included in the online protocol: https://zebrafish.org/wiki/protocols/cryo.\\ \\ ))).\\ |
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| **3.** Prepare 15 mL Falcon tubes (Falcon 352,096) with an empty Matrix cryovial tube (0.5 mL Matrix Screw Top Storage Tubes, Thermo Scientific, Item #3745-BR or 2 mL Corning vials, Item #430488) functioning as a spacer in each along with the Falcon tube caps so that the tubes are ready to hold samples immediately after the cryovials have been capped.\\ | **3.** Prepare 15 mL Falcon tubes (Falcon 352,096) with an empty Matrix cryovial tube (0.5 mL Matrix Screw Top Storage Tubes, Thermo Scientific, Item #3745-BR or 2 mL Corning vials, Item #430488) functioning as a spacer in each along with the Falcon tube caps so that the tubes are ready to hold samples immediately after the cryovials have been capped.\\ |
| **5.** If you are using a multicapper, prepare it with the appropriate caps already attached before adding cryoprotectant (RMMB) to sperm.\\ | **5.** If you are using a multicapper, prepare it with the appropriate caps already attached before adding cryoprotectant (RMMB) to sperm.\\ |
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| **6.** For each sample, determine the volume of RMMB (RMMB volume = 3 x the sperm volume, see Note((Before use, thaw RMMB aliquots in a water bath or heating block at 45-50 ºC. Raffinose precipitates if the RMMB solution is kept on ice. If precipitation occurs, heat the solution slightly to get it back into solution prior to use. Cool the solution to room temperature before mixing with sperm. Keep RMMB at room temperature after thawing.\\ \\ ))).\\ | **6.** For each sample, determine the volume of RMMB (RMMB volume = 3 x the sperm volume, see Note((Before use, thaw RMMB aliquots in a water bath or heating block at 45-50 ºC. Raffinose precipitates if the RMMB solution is kept on ice. If precipitation occurs, heat the solution slightly to get it back into solution prior to use. Cool the solution to room temperature before mixing with sperm. Keep RMMB at room temperature after thawing.\\ \\ ))).\\ |
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| **7.** Add RMMB to sperm and mix by pipetting.\\ | **7.** Add RMMB to sperm and mix by pipetting.\\ |
| - Bring the final volume to 1000 mL with dH<sub>2</sub>O and check the osmolality (should be approximately 300 mmol/kg).\\ - Filter sterilize the solution and store it at 4 ºC.\\ The resulting SS300 solution will contain 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>2, 1 mM MgSO<sub>4</sub>, 10 mM D-(þ)-Glucose, and 20 mM Tris-Cl (8.0). It is used when the cryopreotective medium already contained skim milk. The milk helps to prevent sperm tails from sticking and tangling and is thought to contain antioxidants that protect against oxidative damage during cryopreservation and thawing.\\ | - Bring the final volume to 1000 mL with dH<sub>2</sub>O and check the osmolality (should be approximately 300 mmol/kg).\\ - Filter sterilize the solution and store it at 4 ºC.\\ The resulting SS300 solution will contain 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>2, 1 mM MgSO<sub>4</sub>, 10 mM D-(þ)-Glucose, and 20 mM Tris-Cl (8.0). It is used when the cryopreotective medium already contained skim milk. The milk helps to prevent sperm tails from sticking and tangling and is thought to contain antioxidants that protect against oxidative damage during cryopreservation and thawing.\\ |
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| **2. To prepare Sperm Solution SS300 with 2 mg/mL Difco Skim Milk** (SS300 + milk),\\ - Add 100 mg Difco Skim Milk to 50 mL SS300 and stir or vortex to dissolve.\\ - Aliquot the solution into microcentrifuge tubes and store frozen at -20 ºC.\\ - Thaw and use at room temperature.\\ | **2. To prepare Sperm Solution SS300 with 2 mg/mL Difco Skim Milk** (SS300 + milk),\\ - Add 100 mg Difco Skim Milk to 50 mL SS300 and stir or vortex to dissolve.\\ - Aliquot the solution into microcentrifuge tubes and store frozen at -20 ºC.\\ - Thaw and use at room temperature.\\ |
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| **3.** Remove the cryovial from the LN<sub>2</sub> and quickly open the cap to vent any LN<sub>2</sub> in the vial (see Note((Before squeezing females, sperm samples can be retrieved from the LN2 freezer and maintained | **3.** Remove the cryovial from the LN<sub>2</sub> and quickly open the cap to vent any LN<sub>2</sub> in the vial (see Note((Before squeezing females, sperm samples can be retrieved from the LN2 freezer and maintained |
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| ==== Protocol Notes ==== | ==== Protocol Notes == |
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