The Mouse NaÃ¯ve CD4 T Cells Isolation Kit has been developed for the isolation of untouched naive CD4 T helper cells from suspensions of mouse spleen cells or lymph node cells. Creative Diagnostics offers Mouse NaÃ¯ve CD4 T Cells Isolation for your research. \n
RPMI+: RPMI 1640 with 10% FBS.
ACK buffer: 150 mM NH4Cl, 10 mM KHCO3 and 500 μM EDTA, sterile-filtered.
FACS buffer: PBS with 1 mM EDTA (pH = 8) and 1% BSA, sterile-filtered.
Prepare for cell suspension
1. Isolate spleen (or lymph nodes) from 5- to 10-week-old mice, and place in 5 mL of
RPMI+ in 6 cm dish.
2. Cover the tissue with a 2×2 square of sterile nylon mesh, gently smash the tissue
against the mesh using the thumb side of a 5-mL syringe plunger.
3. Filtrate cell suspension with 40 μm cell strainer, wash the dish and cell strainer
with 5 mL PRMI+, and collect cell suspension in a 15 mL tube.
4. Invert on ice with a few times, centrifuge the cells at 800 rpm for 5 min at 4°C,
5. Add 2 mL ACK buffer per spleen, gently suspend for 1 min to lyse erythrocytes, and
add 10 mL of RPMI+.
Note: For lymph node cells, do not require erythrocyte lysis.
6. Centrifuge at 800 rpm for 5 min at 4°C, remove supernatant.
7. Resuspend with 10 mL RPMI+ buffer, filtrate cells into a new 15 mL tube