I am titering anti-mouse CD3 FITC, PE, and APC antibodies for flow cytometry. CD3 is a T-cell marker, but is not expressed until later in T cell development, so its expression is not well represented in the thymus where we normally titer other T cell markers like CD4 and CD8.
Hi Simone
You can use the spleen. It's easy to isolate and has a lot of T cells.
Hi Simone
You can use the spleen. It's easy to isolate and has a lot of T cells.
I agree with Andre. Go with the spleen - there are tons of cells and they are very easy to obtain.
Good Luck!
G
+1 for mouse splenocytes.
1 Receive mouse spleens.
2 Prepare sterile sieves and cell culture plates.
3 Transfer tissue to a sieve over a fresh cell culture plate with 5 ml media.
4 Make a single cell suspension by gently pushing spleen through the sieve using the plunger of a syringe. Flush the sieve with approx. 5 ml of DM7.
5 Further disintegrate tissue by aspirating through a 23-26g needle.
6 Transfer resulting material into 50 ml tube.
7 Add TAC to 25 ml (approx. 1/5 dilution) and mix well.
8 Incubate at 37°C for 10 minutes.
9 Centrifuge for 10 min at 200xg (~1000 rpm) and room temperature.
10 Aspirate supernatant and resuspend in a final volume of 30 ml PBS.
11 Filter cell suspension through a cell strainer into new (2nd) 50 ml tube.
12 Centrifuge for 10 min at 200xg and room temperature.
13 Aspirate supernatant and resuspend in a final volume of 3 ml PBS.
14 Mix well and transfer 20 μl into counting slide.
15 Perform cell count using cell counter and record counts in table (below)
16 Fill tubes up to 30 ml with PBS
17 Centrifuge for 10 min at 200xg and room temperature.
18 Aspirate supernatant and resuspend at 1x107 cells per ml in DM7.
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