mouse splenocyte preparation - (Oct/16/2000 )
I have prepared spenocyte suspensions/cultures before for proliferation assays using nylon mesh to remove connective tissues and other debris. However, I no longer have the supplier's name for that nylon mesh, and I don't have the specifications for that mesh (filtration size, etc.). Can anyone tell me if they know what I mean? If you haven't used nylon mesh for preparing mouse splenocytes for culture and subsequent assays, what have you used? I'll try that too.
I do not use any mesh for splenocyte preparation. After the spleen has been mashed properly, I leave the debris and carefully aspirate the cell suspension for further process. I hope it should serve the purpose.Best wishesSunita
I use a Falcon 70um nylon mesh. It is product number 2350. If you can't get those, try a 230um steel mesh cup, crushing the spleen through this first and then take the suspension and further filter it through a 70um nylon mesh attached to a test tube.
I use to isolate mouse splenocyte by a very simple way.1/ Cut mouse spleen into several small pices. Then gine spleen pices in metal mesh cup (if you done have one, you can grine spleen bettwen 2 microscope glass slides). 2/Remove all big connective tissue and debris. Collect cell suspension in 15ml facoll tube anf leave the tube stand up about 3-5 mine. The remain debris will come down to the bottom oh the tube.3/ Collect cell suspension to other tube, erythocyte hemolysis, and use for cell proliferation test.
This is how I isolate spleen cells:
2.12.3 Preparation of Spleen Cells
Adult murine spleen cells were used for these applications. The spleens were removed and collected in a sterile wire sieve over a Petri dish half filled with media. The spleens were gently pressed through the sieve using a rubber policeman (which is the plunger in a syringe, usually a 3-5 ml syringer plunger is great), all the while gently applying fresh media on the spleen to keep the cells moist and add transfer through the sieve. The sieve was rinsed over the Petri dish with additional media to remove all remaining cells. The disaggregated cells were transferred to a fresh 30 ml tube (Filtrona) and centrifuged at 2,000 rpm for 5 mins (any tube size will do, we used Filtronas because they were the ideal size for our contrifuge). The cell pellet was resuspended in 10 ml aMEM + 10 % FBS (or which media you are planning to use) and counted.
Hope that helps
BD has 80 um meshes for removal of large debris.
I use 70 µm Cell Strainer for the same propose. you can use that as well.
link attached show thecnical information.
good luck with your experiment
I've used nylon mesh that was very similar to your discription, I bought it through BDH (Merk) but it was supplied by them from an external supplier in the USA. Apologies if thats not much help. However, there are two other very easy ways to prep splenocytes in a sterile manner....
1. Place a 70micron cell sieve on top of a 50ml Falcon tube (containing 10ml medium) and wet the sieve with a little medium. Then place the spleen into the sieve and use the plunger (taken from the inside of a fresh 1ml plastic syringe) to mash the spleen through the gauze, hey presto...splenocytes!
2. The ultrafast method. Autoclave glass slides with frosted ends on them (BDH Merk cat no. 406/0184/04, 50 per box). Place a few ml of medium into a petri dish and use this to carefully wet the frosted ends of the microscope slides. Place the spleen between the wetted ends and gently apply pressure in round circular movements, the spleen should empty quite easily into the surroudning medium. Take the resulting suspension up and down a few times with a P1000 Gilson and, hey presto...single cell suspension of slenocytes in under 5mins.
i was once lost too, hope this helps...
I always use the Mesh from SEFAR company NYTAL ASTM 100-149
Otherwise you can simply use two glass slides and smash them in a circular motion one onto the other with the spleen in between.
I'm working on mouse spleen DCs. I have a BIG problem with splenocytes : I have always a lot, a lot of died cells in my suspensions. after an overnight culture, about 50% are dead. Have you got any solutions for this ? did you already have this problem ?
thanks a lot