Pcr based mycoplasma detection kit

Human embryonic kidney 293T cells (ATCC) and TZM-bl cells (NIH AIDS Reagent Program) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% certified fetal bovine serum, FBS (VivaCell, Shanghai, China). HIV-1 latently infected Jurkat T cells J-Lat A10.6 (J-Lat, NIH AIDS Reagent Program) were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) with 10% FBS. All cell lines were tested mycoplasma-free periodically by a PCR-based mycoplasma detection kit (Beyotime). Healthy human donors’ peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat as previously described. Naive CD4 + T cells were isolated from PBMCs by MACS microbead-negative sorting and the naive CD4 + T-cell isolation kit (Miltenyi Biotec). Primary CD4 + T-cells were maintained in RPMI-1640 with 10% FBS containing 30 U/mL Recombinant human interleukin-2 (IL-2, Sino Biological Inc.).

Human glioblastoma cell line LN229 was purchased from American Type Culture Collection (ATCC). Other human glioblastoma cell lines U251 and U87 were purchased from Procell (Wuhan, China). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37 °C and 5% CO₂. All cell lines were cultured for less than 6 months following resuscitation and were confirmed mycoplasma-free by using the PCR-based mycoplasma detection kit (cat#C0301S, Beyotime-Biotechnology, China).

Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and tested for mycoplasma contamination periodically by a PCR-based mycoplasma detection kit (Beyotime Biotec).
For immunoblot analysis, the following antibodies were used: Mouse anti-Flag (sc-7392, Santa Cruz Biotechnology), Rabbit anti-HA (H6908, Sigma-Aldrich). Rabbit anti-Myc (16286-1), anti-V5 (14440-1), Mouse anti-GFP (66002-1), Mouse anti-GAPDH (60004-1), Mouse anti-Tubulin (66031-1), goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were from Proteintech. Rabbit anti-Flag (14793S), anti-HA (3724S), anti-p65 (8284S), anti-IRF3 (11904T) were purchased from Cell Signaling Technology. Rabbit anti-STAT1 (AF0288) and anti-LaminB1 (AF5222) were from Beyotime Biotec. Mouse anti-HA (201113) and anti-β-Actin (200068-8F10) antibodies were purchased from Zen-Bioscience.