Dulbecco s phosphate buffer saline dpbs

The following materials were used as received: Human colon adenocarcinoma cell line (HT-29), human breast cancer cell line (MDA-MB-231) and human ovarian cancer cell line (SKOV-3) were purchased from ATCC (Manassas, VA, USA). RPMI-1640, Dulbecco’s Modified Eagle’s Medium (DMEM), McCoy’s 5A media, trypsin-EDTA (0.05% trypsin, 0.53 nM EDTA) and Dulbecco’s Phosphate Buffer Saline (DPBS) were from Mediatech-Corning (Manassas, VA, USA). Fetal Bovine Serum (FBS) was from Rocky Mountain Biologicals (Missoula, MT, USA), RIPA buffer was from Amresco (Solon, OH, USA). 3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) was purchased from VWR International (West Chester, PA, USA). Acetazolamide was purchased form Sigma-Aldrich (St. Louis, MO, USA). The ureido-sulfonamide CAIs were synthesized as previously described [57 (link),67 (link)].

Sprague-Dawley rats with hSYN or CMV-promoted GFP AAV2/9 injection in the sciatic nerve were used for electrophysiology experiments. Rats were euthanized by cervical dislocation, the sciatic nerves were immediately immersed into ice cold Dulbecco’s Phosphate-Buffer Saline (DPBS, Corning). The dissected sciatic nerve was cut from the DRG to the end of the sciatic nerve containing the trifurcation among tibial sural and peroneal branches. And then we carefully peeled away the epineural sheath in cold PBS. Collected sciatic nerves were cut into pieces under a fluorescent microscope. The GFP-positive piece was picked up and incubated in Hibernate A (BrainBits) containing papain (100 U; Sigma) at 37°C for 2 h with intermittent flicking. After the removal of enzymes, the collected pieces were trypsinized for 20 min at 37°C. The tissue was triturated into individual cell suspensions by a 1 ml pipette. Enzymes and cellular debris were removed with multiple rounds (three times) of mild centrifugation at 1000 g and washing with Hibernate A minus Ca²⁺ and Mg²⁺ (BrainBits). The individual cell suspension was plated onto a glass bottom plate.

Primary CD4⁺ T cells from healthy human donors were isolated from peripheral blood leukopaks (STEMCELL Technologies). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Cytiva) centrifugation, followed by CD4⁺ T cell isolation using an EasySep Human CD4⁺ T cell Isolation Kit according to manufacturer’s instructions (STEMCELL Technologies). Cells were resuspended at a density of 2.5×10⁶ cells/mL in complete Roswell Park Memorial Institute (RPMI) media consisting of RPMI-1640 (Corning) media with 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Corning), 50 μg/mL penicillin/streptomycin (Corning), 5 mM sodium pyruvate (Corning), and 10% fetal bovine serum (Gibco) and supplemented with 20 IU/mL IL-2 (Miltenyi Biotec) immediately prior to use. Cells were plated 500 μL per well in stimulation plates made by coating 48 well plates with 20 mg/mL anti-CD3 antibody (UCHT1, Tonbo Biosciences) diluted in Dulbecco’s Phosphate Buffer Saline (DPBS, Corning) overnight at 4°C, with soluble anti-CD28 (CD28.2, Tonbo Biosciences) added at 5 mg/mL to the cell suspension at the time of plating. Cells were stimulated for 72 h in a cell culture incubator at 37°C with 5% CO₂ prior to CRISPR-Cas9 editing.