NSET – Helpful Hints & Tips
The following are tips and suggestions from our scientists/technicians/customers which have proved helpful.
- Read all “Hints & Tips” and the NSET instruction insert carefully before beginning your NSET trials. The instruction insert can be found in each box of NSET.
- Prior to actual experiments, practice the NSET technique on 2.5 dpc pseudopregnant mice without embryos.
- NSET is designed to fit snugly on a Rainin Classic PR2, 0.1-2µl or Gilson Pipetman P2, 0.2-2µl pipette for loading embryos and precise measurement of media into the tip of the device.
- Scientists highly recommend that you only use mice that are not sedated. This makes it easier to get the mouse in a natural position to find and enter the cervix.
- The NSET device is only able to pass the cervix during certain phases of estrus and at 2.5 dpc in pseudopregnant mice. Therefore we strongly recommend the use of 2.5 dpc pseudopregnant mice (after the plug has fallen out) for training purposes and embryo transfers using the NSET device.
- CD1 mice are highly recommend using this strain for your pseudopregnant recipient. We suggest using mice that weigh ≥26g and at least 60 days old.
- Embryos should be incubated to blastocyst stage (3.5 days) since the device transfers them to one of the uterine horns and not the oviduct. Some end users have been successful using morula embryos.
- Select a media you have used which gives you the best success in incubating your embryos. For example, use KSOM medium (or other desired culture media) and transfer 12 to 20 embryos.
- Gooseneck lighting is highly recommended to help locate the cervical opening.
- Scientists suggest using conscious, calm, and unagitated mice. The female mouse in the video on our website is not sedated.
- It is relatively easy to keep the female still and reduce squirming by placing the mouse on top of the cage so she can grasp the wires. Use the holding technique as demonstrated in the video. (Watch the NSET video)
- The use of lubricants is not recommended. You may use sterile water or culture media to moisten the specula then shake off excess before insertion into the vagina.
- Insert small speculum first, remove, then insert larger speculum. The mouse will innately push the speculum out a little. Gently press it back in place so the NSET device can pass through the cervix and into one of the uterine horns.
- Be patient and do not apply too much pressure when finding and penetrating the cervix. This could cause tissue damage and will likely bend the NSET device making it nearly impossible to use. If the first attempt to insert the NSET is not in the correct location, gently reposition the device and repeat.
- Embryo loss may occur if tip gets bent due to too much pressure asserted while finding the cervical opening. Again, gentle repeated attempts are pertinent to NSET success.
- You will know the device is properly inserted through the cervix into the uterus when the hub of the NSET device touches the end of the larger speculum.
- To expel your embryos press the pipette plunger all the way down. Do not release plunger.
- Immediately remove NSET without releasing pipette plunger. If plunger is released prior to removal, some embryos could be pulled back into the tip.
- Inspection of the NSET tip under a microscope after use is good practice. The new clear tip NSET allows visualization inside the tip.
Keep in mind this device is designed for a one-time use only. Repeated use will clog the NSET tip with tissue which could lead to unfavourable results. Please make sure to read the NSET instruction sheet thoroughly before use.
Studies have shown comparable efficiency to surgery. Please let us know of your trials with the device and feel free to contact us with any questions. We are always willing to help. The NSET device paper can be found in BioTechniques’ November 2009 issue. Thank you again for your interest in NSET and we look forward to hearing from you.