Stempro 34 nutrient supplement

The human MC line LAD2 was a gift from Dr. A. S. Kirshenbaum (National Institutes of Health, Bethesda, MD) [16 (link)]. Cells were cultured at 37 °C in 5% CO₂ and passaged weekly to maintain a 500,000 cells/mL density. Dedicated medium consisted of StemPro-34 serum-free medium (Gibco) supplemented with StemPro-34 Nutrient Supplement (Gibco), 2 mM l-glutamine (Sigma-Aldrich), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), and 100 ng/mL recombinant human SCF (R&D Systems).

The neoplastic huMC lines HMC‐1.1 (KIT V560G) and HMC‐1.2 (KIT V560G, KIT D816V) were kindly provided by Dr. JH Butterfield (Mayo Clinic, Rochester, MN, USA) (Butterfield et al., 1988 (link)). HMC‐1 cells were maintained in a humidified incubator at 37°C and 5% CO₂ in T75 flasks in Iscove's modified Dulbecco's medium (IMDM, Corning) supplemented with 2 mM L‐Glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (Corning) and 10% (v/v) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific). Cells were passaged every 3–4 days based on cell density. The LAD2 huMC line was maintained in serum‐free StemPro‐34 media supplemented with StemPro‐34 nutrient supplement (Gibco), 2 mM L‐Glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (Corning) and 100 ng/ml recombinant human SCF (R&D Systems), as described (Kirshenbaum et al., 2003 (link)). Hemi‐depletions were performed weekly. Cell counts and viability (using acridine orange/propidium iodide stain, Logos Biosystems) were assessed on a Luna‐FL automated brightfield and dual fluorescence cell counter (Logos Biosystems). Cell treatment with 10 μM GW4869 (5 mM stock solution in DMSO; Sigma‐Aldrich), DMSO (vehicle control; Sigma‐Aldrich) or 10 μM amitriptyline (10 mM stock solution in water; Sigma‐Aldrich) was performed for 16 h.

Jurkat (ATCC, Manassas, VA, USA), Jurkat NFAT (BPS Biosciences, San Diego, CA, USA), THP-1 NF_KB (BPS Biosciences), and U937 (ATCC) cell lines were maintained in RPMI 1640 medium (Clontech, San Jose, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA). RBL2H3 (ATCC) and HEK293 (ATCC) cell lines were maintained in DMEM medium (Clontech) supplemented with 10% fetal bovine serum. The Luva cell line (Kerafast, Boston, MA, USA) was maintained in StemPro-34 SFM medium supplemented with StemPro-34 Nutrient supplement and 2 m m L-glutamine (Gibco). All cells were cultured at 37°C, 5% CO₂, and 95% humidity.

Three human B- lymphoma cell lines were used in this study, WSU-WM [12 (link)], WSU-FSCCL [13 (link)], and WSU-DLCL2 [14 (link)]. The WSU-WM cell line is an IgM-secreting lymphoplasmacytic lymphoma established from a patient with Waldenström’s macroglobulinemia (WM). The WSU-FSCCL cell line was established from a patient with follicular small cleaved cell lymphoma (FSCCL) which is equivalent to follicular lymphoma, grade 1 by WHO classification [15 (link)]. The WSU-DLCL2 represents an aggressive diffuse large cell lymphoma (DLCL). All cell lines were established by the Lymphoma Research Laboratory at Wayne State University (WSU), Epstein-Barr virus (EBV)-negative, well characterized and authenticated. Cells are maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and antibiotics as previously described [16 (link)]. CD34⁺ cells were also cultured in StemPro^®-34 SFM medium. This is a serum-free medium (SFM) containing StemPro^®- 34 nutrient supplement (Gibco by Life Technologies) specifically formulated to support the development of human hematopoietic cells in culture.

All cell types described below were incubated at 37°C in a 5% CO₂ humidified atmosphere, cells were grown in tissue culture‐treated flasks.
LAD2 were cultured in StemPro‐34 media supplemented with StemPro‐34 nutrient supplement and 2 mmol/L L‐glutamine (all Gibco Life Technologies) in addition to 100 ng/mL recombinant human stem cell factor (rhSCF) (R&D systems). Cells were passaged weekly; media was added to maintain a density of 400,000–500,000 cells/mL.
For data presented in Figure 5, human embryonic kidney cells stably expressing human TRPC6 (HEK‐ TRPC6) were cultured in DMEM containing 10% FCS and 400 μg/mL geneticin (Gibco) to select for TRPC6 expression.
For data presented in Figure S5, human and rat TRPC3 and TRPC6 channels were heterologously expressed in human embryonic kidney 293 (HEK293) cells, using BacMam transduction. All human and rat TRPC3 and TRPC6 BacMam reagents were generated at GlaxoSmithKline (King of Prussia, PA). HEK293 cells were grown in 6‐well culture dishes, using DMEM F‐12 medium supplemented with 10% FBS and 1% Pen/Strep. 6–12% TRPC3 or TRPC6 BacMam virus were added to 6‐well culture dishes 24~48 h before experiments. HEK 293 cells were detached from the culture dish using trypsin solution (0.25% trypsin + 0.1% EDTA) and stored in the culture medium at room temperature for patch‐clamp experiments within 5 h.

The purchased chemicals and kits from respective company used were Propidium Iodide (PI) (Calbiochem, #537059), Ribonuclease A (RNase A, Biotech, #9001-99-4.), ethanol (Sigma Aldrich, #1009832500), PBS (Sigma Aldrich, #P4417), fetal bovine serum (FBS) (Gibco, USA), antibiotic (Himedia, #A018), Iscove's Modified Dulbecco's Medium (IMDM) (Sigma Aldrich, #I7633), N (Calbiochem, #565851), Human S (Sigma Aldrich, #57901), BrdU colorimetric cell proliferation kit (Calbiochem, #JA1599), 5(6)-carboxyfluorescein diacetate N succinimidyl ester (CFDA-SE) (Sigma Aldrich, #21888), PrestoBlue (Invitrogen, #A13261), Paraformaldehyde (PFA) (Sigma Aldrich, #P6148), Ki-67 mouse monoclonal IgG antibody (SantaCruz, #23900), Goat Anti-mouse IgG FITC Conjugated (Sigma Aldrich, #F5387), Bovine Serum Albumin (BSA) (AMRESCO, #0332), sodium azide (HiMedia, #GRM1038), saponin (Calbiochem, #558255), StemPro-34 nutrient supplement (Gibco, #10641), CyQuant cell proliferation assay kit (Invitrogen, #35011), Anti-CD117 antibody (c-Kit-PE, Millipore, #10482), Anti-Phosho c-Kit (pY568 and pY570) antibody (Abcam, #ab5616), Rabbit monoclonal Anti-SHP-1/2 (Millipore, #04742), Goat Anti-rabbit IgG-R-PE (Invitrogen, #PZ771MP), Mouse Anti-HePTP (PTPN7) IgG2b_κ (Millipore, #04278), Anti-mouse IgG2b FITC (Sigma Aldrich, #SAB3701184).

Flow sorted primary mast cells were maintained in human mast cell culture media (StemPro^TM-34 Serum-Free media (Gibco), StemPro-34 Nutrient Supplement (Gibco), L-Glutamine (2 mM) (Gibco), Penicillin (100 U/mL)/Streptomycin (100 µg/mL) (Gibco), recombinant human stem cell factor (100 ng/mL) (PeproTech), recombinant human Interleukin 6 (100 ng/mL) (PeproTech) and Recombinant human Interleukin-3 (30 ng/mL) (PeproTech)) at 37 °C in 5% CO₂, with media changes every 5–7 days [22 (link)]. Primary mast cell numbers were determined with TC-20^TM Automated Cell Counter system (Bio-Rad) according to manufacturer’s protocol at every media change. Growth curves for primary mast cells are shown in Figure S3a.