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protocols:step-by-step_cryopreservation_protocol [2021/10/29 19:42] zoltan created | protocols:step-by-step_cryopreservation_protocol [2023/04/27 09:38] ron created | ||
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- | ====== Step-by-Step Pooling, Freezing, | + | ====== Step-by-Step |
+ | {{ : | ||
===== OVERVIEW ===== | ===== OVERVIEW ===== | ||
- | {{ :cryo:cryopreservation_thawing_overview.png?300|}} | + | {{ :protocols:zirc_wiki_step_by_step_cryo_protocol_figure.04.24.2023.jpg?200|}}\\ |
1. Collect sperm and pool in extender E400G\\ | 1. Collect sperm and pool in extender E400G\\ | ||
2. Measure cell density\\ | 2. Measure cell density\\ | ||
- | 3. Dilute with E400G. Target: | + | 3. Dilute with E400G. Target: 4x10< |
4. Add RMMB Cryoprotectant solution (1V sperm + 3V RMMB)\\ | 4. Add RMMB Cryoprotectant solution (1V sperm + 3V RMMB)\\ | ||
5. Aliquot into cryovials (20 µl) and cap\\ | 5. Aliquot into cryovials (20 µl) and cap\\ | ||
- | 6. Freeze\\ | + | 6. Freeze |
7. Store in LN< | 7. Store in LN< | ||
- | 8. Squeeze eggs, pool or split clutches as needed; store max 10 min in humid chamber\\ | + | 8. Squeeze eggs, pool clutches as needed; store max. 10 minutes |
9. Retrieve & thaw sample\\ | 9. Retrieve & thaw sample\\ | ||
- | 10. Add SS300 with or without milk\\ | + | 10. Add SS300\\ |
- | 11. Activate Sperm\\ | + | 11. Transfer to eggs\\ |
- | 12. Fertilize\\ | + | 12. Activate to fertilize\\ |
13. Do **NOT** swirl or mix fertilized eggs/dish. Wait 2 minutes\\ | 13. Do **NOT** swirl or mix fertilized eggs/dish. Wait 2 minutes\\ | ||
14. Add embryo medium; rear embryos\\ | 14. Add embryo medium; rear embryos\\ | ||
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===== A. Collection and pooling of sperm ===== | ===== A. Collection and pooling of sperm ===== | ||
- | **1.** Prepare a 0.5 mL microcentrifuge tube with E400G for each fish line (see Note((A few general guidelines for selecting the starting volume of E400G into which sperm is collected are provided. The use of an appropriate starting volume will ensure that the sperm concentration is as optimal as possible in the resulting samples without activating the cells. | + | **1.** Prepare a 0.5 mL microcentrifuge tube with E400 for each fish line (see Note((A few general guidelines for selecting the starting volume of E400 into which sperm is collected are provided. The use of an appropriate starting volume will ensure that the sperm concentration is as optimal as possible in the resulting samples without activating the cells. |
- | The estimated | + | The starting volume of E400 depends on the number of available males (short-fin fish typically give more sperm than long-fin fish). To calculate a conservative starting volume of E400, use the following equations.\\ |
- Short-fin fish: (# males-2) x 5 µl E400G\\ | - Short-fin fish: (# males-2) x 5 µl E400G\\ | ||
and\\ | and\\ | ||
- | - Long-fin fish: (# males-4) x 5 µl E400G\\ \\ )) to estimate | + | - Long-fin fish: (# males-4) x 5 µl E400G\\ \\ )) to estimate |
- Add dry components first, stir to dissolve, add liquid components, and stir.\\ | - Add dry components first, stir to dissolve, add liquid components, and stir.\\ | ||
- Adjust the pH to 7.9 with 5 M KOH and bring the final volume to 1000 mL with dH< | - Adjust the pH to 7.9 with 5 M KOH and bring the final volume to 1000 mL with dH< | ||
- | It is advisable to check the osmolality of E400, which should be close to 400 mmol/kg. The solution should be filter sterilized and stored at 4 ºC. The resulting E400G contains 130 mM KCl, 50 mM NaCl, 2 mM CaCl< | + | It is advisable to check the osmolality of E400, which should be close to 400 mmol/kg. The solution should be filter sterilized and stored at 4 ºC. The resulting E400G contains 130 mM KCl, 50 mM NaCl, 2 mM CaCl< |
- | **2.** Keep E400G and the collected sperm at room temperature | + | **2.** Keep E400 and the collected sperm on ice throughout the collection and prefreezing procedures.\\ |
- | **3.** Sedate or preanesthetize males as needed for at least 10 min before the procedure (see Note((Preanesthesia/ | + | **3.** Sedate or preanesthetize males as needed for at least 10 min before the procedure (see Note((Preanesthesia/ |
**4.** For anesthesia, dilute 4.2 mL tricaine stock solution in 100 mL fish water (168 mg/L). Anesthetize two to three males in 168 mg/L MS-222.\\ | **4.** For anesthesia, dilute 4.2 mL tricaine stock solution in 100 mL fish water (168 mg/L). Anesthetize two to three males in 168 mg/L MS-222.\\ | ||
**5.** Briefly rinse a male in phosphate buffered saline (PBS) isotonic fish rinse, dry it by rolling gently on a soft and absorbent paper towel (see Note((Eggs and sperm are activated by water, and it is virtually impossible to dry fish completely. Therefore, it is important to rinse anesthetized males and females with isotonic PBS solution before gamete collection to avoid premature activation of sperm or unfertilized eggs. | **5.** Briefly rinse a male in phosphate buffered saline (PBS) isotonic fish rinse, dry it by rolling gently on a soft and absorbent paper towel (see Note((Eggs and sperm are activated by water, and it is virtually impossible to dry fish completely. Therefore, it is important to rinse anesthetized males and females with isotonic PBS solution before gamete collection to avoid premature activation of sperm or unfertilized eggs. | ||
- | \\ \\ ))), and place it belly up in a dampened sponge/foam holder (in a 35 * 10 mm Petri dish). The isotonic PBS fish rinse is prepared from PBS (pH 7.4) powder packets (Sigma #P3813) that are dissolved in 870 mL dH< | + | \\ \\ ))), and place it belly up in a dampened sponge/foam holder (in a 35 x 10 mm Petri dish). The isotonic PBS fish rinse is prepared from PBS (pH 7.4) powder packets (Sigma #P3813) that are dissolved in 870 mL dH< |
**6.** Place the end of a calibrated 10 mL borosilicate glass microcapillary (Drummond #2-000-010) on the urogenital opening.\\ | **6.** Place the end of a calibrated 10 mL borosilicate glass microcapillary (Drummond #2-000-010) on the urogenital opening.\\ | ||
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**7.** Use rubber-tipped Millipore forceps (Millipore # | **7.** Use rubber-tipped Millipore forceps (Millipore # | ||
- | **8.** Expel sperm immediately into the E400G solution in the microcentrifuge collection tube.\\ | + | **8.** Expel sperm immediately into the E400 solution in the microcentrifuge collection tube.\\ |
**9.** Transfer fish into fresh system water for recovery from anesthesia.\\ | **9.** Transfer fish into fresh system water for recovery from anesthesia.\\ | ||
- | **10.** Continue collecting sperm from all males from the same family/ | + | **10.** Continue collecting sperm from all males from the same family/ |
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===== B. Estimation of sperm cell densities and sample dilution ===== | ===== B. Estimation of sperm cell densities and sample dilution ===== | ||
- | At ZIRC, we use a NanoDrop 2000 spectrophotometer | + | We use the ZIRC NanoDrop 2000 Calibration Curve and Sperm Density Calculator |
cell density. There are two key benefits to adjusting cell densities: | cell density. There are two key benefits to adjusting cell densities: | ||
and IVF protocol (PDF) and Excel sperm density calculator files from the ZIRC website: https:// | and IVF protocol (PDF) and Excel sperm density calculator files from the ZIRC website: https:// | ||
to the A< | to the A< | ||
- | **1.** Estimate the volume of pooled sperm in E400G in the microcentrifuge tube for each line using a Pipetman. Draw the sperm into the tip and adjust the pipette volume until all of the solution fills the tip. Record the estimated volume for later use. While measuring, gently pipette the sperm to mix completely.\\ | + | **1.** Estimate the volume of pooled sperm in E400 in the microcentrifuge tube for each line using a Pipetman. Draw the sperm into the tip and adjust the pipette volume until all of the solution fills the tip. Record the estimated volume for later use. While measuring, gently pipette the sperm to mix completely.\\ |
- | **2.** Prepare a 1:10 dilution of the sperm solution in E400G for a density measurement. If the collected sample appears less opaque (i.e., less concentrated), | + | **2.** Prepare a 1:10 dilution of the sperm solution in E400 for a density measurement. If the collected sample appears less opaque (i.e., less concentrated), |
**3.** Mix the diluted sample by flicking the tube and keep the tube at room temperature. Optional: Use separate (colored) microcentrifuge tubes for each line (see Note((Using a designated colored microcentrifuge tube for the NanoDrop dilution makes it easily distinguishable from samples of other stocks.\\ \\ ))).\\ | **3.** Mix the diluted sample by flicking the tube and keep the tube at room temperature. Optional: Use separate (colored) microcentrifuge tubes for each line (see Note((Using a designated colored microcentrifuge tube for the NanoDrop dilution makes it easily distinguishable from samples of other stocks.\\ \\ ))).\\ | ||
- | **4.** Calibrate A400 absorption of a blank E400G sample using the NanoDrop’s Cell Cultures menu. Set the Absorbance Cursor to 400 nm and add 1.5 mL E400G to the spectrophotometer. Read the E400G blank sample to verify that it has set the instrument’s readings close to 0. If a blank sample reads higher than 0.005, recalibrate with a fresh 1.5 mL aliquot of E400G. When prompted, save the NanoDrop data with the date of the freeze event and line name(s).\\ | + | **4.** Calibrate A400 absorption of a blank E400G sample using the NanoDrop’s Cell Cultures menu. Set the Absorbance Cursor to 400 nm and add 1.5 µL E400 to the spectrophotometer. Read the E400 blank sample to verify that it has set the instrument’s readings close to 0. If a blank sample reads higher than 0.004, repeat |
**5.** To measure the absorption of a sample, enter the sample ID on the Measure Cell Cultures page.\\ | **5.** To measure the absorption of a sample, enter the sample ID on the Measure Cell Cultures page.\\ | ||
- | **6.** Mix the sample well using a vortex mixer set at intermediate speed (~1300 rpm).\\ | + | **6.** Mix the sample well by flicking or using a vortex mixer set at intermediate speed (~1300 rpm).\\ |
- | **7.** Immediately load 1.5 mL of the diluted sperm and read the cursor absorbance (A< | + | **7.** Immediately load 1.5 µL of the diluted sperm and read the cursor absorbance (A< |
**8.** Measure each sample three to five times and calculate the average A400 for all samples. A minimum of three successful readings is optimal.\\ | **8.** Measure each sample three to five times and calculate the average A400 for all samples. A minimum of three successful readings is optimal.\\ | ||
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**9.** Clean the NanoDrop using a clean Kimwipe and deionized water. Wipe the top arm (mirror) and the bottom lens with a moistened Kimwipe and then dry completely with a Kimwipe before closing the arm.\\ | **9.** Clean the NanoDrop using a clean Kimwipe and deionized water. Wipe the top arm (mirror) and the bottom lens with a moistened Kimwipe and then dry completely with a Kimwipe before closing the arm.\\ | ||
- | **10.** Calculate the cell density for each sample with the averaged A400 using the Excel cell density calculator.\\ | + | **10.** Calculate the cell density for each sample with the averaged A400 using the ZIRC NanoDrop 2000 Calibration Curve and Sperm Density Calculator or similar lab specific calibration curve.\\ |
- | **11.** Dilute the sperm with E400G according to the desired number of samples or sperm concentration. Optimal sperm cell densities should be between 4.0*10< | + | **11.** Dilute the sperm with E400 according to the desired number of samples or sperm concentration. Optimal sperm cell densities should be between 4.0*10< |
samples with 2.0*10< | samples with 2.0*10< | ||
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\\ | \\ | ||
- | ===== C. Pooled sperm freezing ===== | + | ===== C. Sperm freezing ===== |
**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< | **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< | ||
- | - 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, | + | - 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, |
- | **2.** Fill a Styrofoam container or cooler (z30 cm 30 cm) with powdered dry ice made from liquid CO2 (Carmichael et al., 2009) (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/ | + | **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:// |
**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.\\ | ||
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**5.** If you are using a multicapper, | **5.** If you are using a multicapper, | ||
- | **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 diluted sperm and RMMB at room temperature.\\ \\ ))).\\ | + | **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 |
**7.** Add RMMB to sperm and mix by pipetting.\\ | **7.** Add RMMB to sperm and mix by pipetting.\\ | ||
- | **8.** Immediately aliquot 20 mL into the prelabeled 0.5 mL cryovials.\\ | + | **8.** Immediately aliquot 20 µL into the prelabeled 0.5 mL cryovials.\\ |
**9.** Without delay, cap the cryovials (use an automated capper for rows of eight tubes, if available) and place the cryovials into the 15 mL conical tubes (containing a Matrix cryovial spacer).\\ | **9.** Without delay, cap the cryovials (use an automated capper for rows of eight tubes, if available) and place the cryovials into the 15 mL conical tubes (containing a Matrix cryovial spacer).\\ | ||
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\\ \\ ))).\\ | \\ \\ ))).\\ | ||
- | **11.** Freeze the samples in dry ice for 20 - 40 min and then quickly transfer them to a cryogenic freezer box submerged in LN< | + | **11.** Freeze the samples in dry ice for 20 - 45 min and then quickly transfer them to a cryogenic freezer box submerged in LN< |
\\ | \\ | ||
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**5.** Dampen fingers with PBS fish rinse.\\ | **5.** Dampen fingers with PBS fish rinse.\\ | ||
- | **6.** Expel eggs by applying gentle (light) finger pressure on the ventral abdomen and move your finger from anterior to posterior. Eggs will be expelled readily if the female is ready (see Note((Good eggs will be golden in color and have little fluid and no opaque or white eggs intermixed. Eggs can be gently moved away from the fish or split into different dishes using a fine water-color | + | **6.** Expel eggs by applying gentle (light) finger pressure on the ventral abdomen and move your finger from anterior to posterior. Eggs will be expelled readily if the female is ready (see Note((Good eggs will be golden in color and have little fluid and no opaque or white eggs intermixed. Eggs can be gently moved away from the fish or split into different dishes using a fine paint brush.\\ \\ ))).\\ |
**7.** Transfer the female to a recovery tank.\\ | **7.** Transfer the female to a recovery tank.\\ | ||
- | **8.** Combine several clutches of eggs if needed by gently moving eggs to another dish with a fine-tipped paint brush dampened with isotonic PBS (see Note((If females do not provide large enough clutches, or to maximize the number of embryos produced by IVF, it is helpful to combine several clutches of eggs. A soft water-color | + | **8.** Combine several clutches of eggs if needed by gently moving eggs to another dish with a fine-tipped paint brush dampened with isotonic PBS (see Note((If females do not provide large enough clutches, or to maximize the number of embryos produced by IVF, it is helpful to combine several clutches of eggs. A soft paint brush is an effective and gentle tool for moving and mixing eggs. Dampen the fingers and paint brush with the isotonic PBS solution before squeezing females or manipulating eggs.\\ \\ ))).\\ |
- | **9.** Maintain pooled eggs in a closed dish in a moisture chamber at room temperature no longer than 5e10 min before IVF.\\ | + | **9.** Maintain pooled eggs in a closed dish in a moisture chamber at room temperature no longer than 5-10 min before IVF.\\ |
\\ | \\ | ||
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- Bring the final volume to 1000 mL with dH< | - Bring the final volume to 1000 mL with dH< | ||
- | **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.\\ |
**3.** Remove the cryovial from the LN< | **3.** Remove the cryovial from the LN< | ||
in a tray of LN2 within a small Styrofoam cooler until eggs are available.\\ \\ ))).\\ | in a tray of LN2 within a small Styrofoam cooler until eggs are available.\\ \\ ))).\\ | ||
- | **4.** Thaw the cryovial in a 38 ºC water bath until the frozen pellet is less than 3 mm in diameter (takes approximately 10 - 15 s.\\ | + | **4.** Thaw the cryovial in a 38 ºC water bath until the frozen pellet is less than 3 mm in diameter (takes approximately 10 - 15 s).\\ |
- | **5.** Immediately add 150 mL room-temperature SS300 solution to the cryovial. If you are thawing sperm that was frozen without milk (see Note((SS300 + milk is used for thawing sperm samples that were frozen with a cryopreservation medium not containing skim milk powder (other protocols). The milk helps to prevent the sperm tails from sticking and tangling and is thought to contain antioxidants that mitigate oxidative damage. \\ \\ ))), add 2 mg/mL Difco Skim Milk (Difco #232100) to the SS300 solution. See Note((For the postthaw motility assessment, a small portion (10=20 µL) of the sperm/SS300 mix can be removed and held in a microcentrifuge tube at room temperature if assessed immediately or on ice if stored for later (see the “Motility assessment” section).\\ \\ )) for an optional postthaw motility assessment.\\ | + | **5.** Immediately add 200 µL room-temperature SS300 solution to the cryovial. If you are thawing sperm that was frozen without milk (see Note((SS300 + milk is used for thawing sperm samples that were frozen with a cryopreservation medium not containing skim milk powder (other protocols). The milk helps to prevent the sperm tails from sticking and tangling and is thought to contain antioxidants that mitigate oxidative damage. \\ \\ ))), add 2 mg/mL Difco Skim Milk (Difco #232100) to the SS300 solution. See Note((For the postthaw motility assessment, a small portion (10-20 µL) of the sperm/SS300 mix can be removed and held in a microcentrifuge tube at room temperature if assessed immediately or on ice if stored for later (see the “Motility assessment” section).\\ \\ )) for an optional postthaw motility assessment.\\ |
\\ | \\ | ||
===== F. In vitro fertilization ===== | ===== F. In vitro fertilization ===== | ||
- | **1.** | + | **1.** |
- | - Gently mix the sperm 1 - 2 times with a micropipette and transfer the sample to the eggs:\\ | + | - Slide the pipette tip sideways along the bottom of the Petri dish from the edge of the pile of eggs into the center.\\ - Expel the sperm into the mass of eggs (not on top of the eggs).\\ |
- | - slide the pipette tip sideways along the bottom of the Petri dish from the edge of the pile of eggs into the center.\\ - Expel the activated | + | |
- | **2.** | + | **2.** |
- | **3.** | + | **3.** |
- | **4.** Determine the fertilization rate 2 - 4 h postfertilization or as soon as cell divisions are recognizable.\\ - Count embryos and remove unfertilized eggs (see Note((A fertilized egg will be visible by the swelling of the first embryonic cell (zygote) and the chorion or the first cell divisions. Because fertilization and the emergence of the first cell and | + | **4.** Do **not** move, mix, or swirl the dish; let it sit completely undisturbed for 2 minutes, then flood the dish with embryo medium.\\ |
- | its division are usually not perfectly synchronized, | + | |
+ | **5.** Determine the fertilization rate 2 - 4 h postfertilization or as soon as cell divisions are recognizable.\\ - Count embryos and remove unfertilized eggs (see Note((A fertilized egg will be visible by the swelling of the first embryonic cell (zygote) and the chorion or the first cell divisions. Because fertilization and the emergence of the first cell and | ||
+ | its division are usually not perfectly synchronized, | ||
\\ | \\ | ||
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**To estimate the prefreeze motility**, | **To estimate the prefreeze motility**, | ||
- | **1.** Place 6 mL dH< | + | **1.** Place 6 µL dH< |
**2.** Add 0.5 - 1 µL of the final sperm dilution (in E400G) to the drop and then mix the drop and spread it quickly with the pipette tip.\\ | **2.** Add 0.5 - 1 µL of the final sperm dilution (in E400G) to the drop and then mix the drop and spread it quickly with the pipette tip.\\ | ||
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**To estimate the postthaw sperm motility**, | **To estimate the postthaw sperm motility**, | ||
- | **2.** add 150 µL of SS300 solution to the thawed sperm, and mix gently.\\ | + | **2.** add 200 µL of SS300 solution to the thawed sperm, and mix gently.\\ |
**3.** Remove 10 - 20 µL of the sample for motility assessment (see Note((At this point, a small portion (10-20 µL) of the sample can be removed and held on ice for motility assessment. The remainder of the sample can be used for IVF as described earlier. It is best to view the motility as soon as possible after thawing, but samples are typically stable on ice for 10-20 min. \\ \\ ))).\\ | **3.** Remove 10 - 20 µL of the sample for motility assessment (see Note((At this point, a small portion (10-20 µL) of the sample can be removed and held on ice for motility assessment. The remainder of the sample can be used for IVF as described earlier. It is best to view the motility as soon as possible after thawing, but samples are typically stable on ice for 10-20 min. \\ \\ ))).\\ | ||
- | **4.** Place 5 mL dH< | + | **4.** Place 5.8 µL dH< |
**5.** Mix briefly with the pipette tip on the slide and observe immediately.\\ | **5.** Mix briefly with the pipette tip on the slide and observe immediately.\\ | ||
\\ | \\ | ||
- | ==== Protocol Notes ==== | + | ==== Protocol Notes == |
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