8 replies to this topic

[anonymous]

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.

Thanks!

[anonymous]

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

[anonymous]

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.

[anonymous]

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.

By 3/

[valkyrie]

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

Dani

[immune platelet]

BD has 80 um meshes for removal of large debris.

[usflucy]

Does anybody use Falcon 40um nylon mesh for splenocytes or thymocytes collection?

[shyarasu]

' date='Oct 18 2001, 10:00 PM said:

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.<p>By 3/

Hi, Does anybody use lipopeptide antigens in immunological reactions? What tests are best suited for them? Can we use MQ water to reconstitute synthetically prepared lipopetides? What about culture medium?

[Sweetsugar70]

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