Stem pro 34 sfm media

StemPro 34 SFM media and nutrient supplement were obtained from Invitrogen Life Technologies (Paisley, UK). Stem cell factor was obtained from R&D systems (Abingdon, UK). Nucleotides, apyrase, PPADS, poly-L-lysine, EGTA, D-glutamic acid, 4-nitrophenyl N-acetyl-β-D-glucosaminide, Trizma HCl, citric acid and HEPES were purchased from Sigma-Aldrich (Poole, Dorset, UK). Physiological salts, sodium hydroxide and glucose were purchased from BDH anachem. Fura-2 AM was purchased from Calbiochem (Merck Chemicals Ltd., Nottingham, UK) or Invitrogen Ltd. (Paisley, UK). Cesium hydroxide was purchased from ICN Biomedicals Inc. (USA). NF 449 was purchased from Tocris Bioscience (Bristol, UK). AZ 11645373 was a generous gift from AstraZeneca R&D (Charnwood, UK).

The following cell lines were used in this study: p53 wild-type/FLT3ITD: MOLM-13 (adult acute myeloid leukemia), MV4-11 (adult acute myeloid leukemia, MOLT-4 (acute lymphoblastic leukemia). p53 wild-type/FLT3 wild-type: ML-2 (adult acute myeloid leukemia), M-07e (adult acute myeloid leukemia). p53-null/mutant: HL-60 (adult acute myeloid leukemia), THP-1 (adult acute myeloid leukemia). They were purchased from ATCC (Manassas, VA) or DSMZ (Braunschweig, Germany) and maintained in RPMI 1640 media supplemented with 20% heat-inactivated fetal bovine serum (FBS). M-07e cells were cultured in RPMI 1640 supplemented with heat-inactivated fetal bovine serum and GM-CSF (10 ng/ml). All cell lines were maintained at low passage and confluence. Short tandem repeats (STRs) were analyzed to confirm cell line identity and mycoplasma infection was excluded by a PCR-based testing. Human bone marrow (BM) CD34 + cells obtained from healthy donors were purchased from StemCell Technologies, Inc. (Vancouver, BC, Canada) and maintained in StemPro-34-SFM media from GIBCO (Waltham, MA). They were maintained for up to 7 days in liquid culture media. Healthy bone marrow donor-derived MSC were collected under MD Anderson Cancer Center Institutional Review Board approval (LAB02-0395). Human BM-derived MSCs were isolated as described previously^{58 (link)}.

Immortalized human mast cell line, LUVA (Kerafast, USA), was used to model lung-resident mast cell response to SARS-CoV-2 infection. We and others (Laidlaw et al., 2011) have shown that LUVA cells express FcεRI and c-Kit similarly to normal human MC. Cells were cultured in StemPro-34 SFM media (Gibco, USA) with L-glutamine (2 mM) and Penicillin-Streptomycin (10 U/ml) in a humidified incubator at 5% CO₂ and 37°C. Cells were maintained at 5x10⁵/ml and passaged up to 15 times; FcεRI and c-Kit expression the cell surface of LUVA cells was maintained throughout passaging.

The LAD2 cell line (a gift from Arnold Kirshenbaum and Dean Metcalfe from the National Institutes of Health, NIAID) was cultured in StemPro-34 SFM media (Life Technologies, Burlington, ON, Canada) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ, USA). Cells were maintained at 1×10⁵ cells/ml at 37°C and 5% CO₂ and periodically tested for the expression of CD117 (eBioscience, Invitrogen Carlsbad, CA, USA), and FcϵRI (eBioscience, Invitrogen Carlsbad, CA, USA) by flow cytometry using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA).

HMC-1.1 and HMC-1.2 human mast cell lines were cultured in Iscove's Modified Dulbecco's Medium (Lonza, Mississauga, ON, Canada) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 1  ×  10⁵ cells/ml at 37°C and 5% CO₂. The LAD2 cell line was cultured in StemPro-34 SFM media (Life Technologies, Burlington, ON, Canada) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ). Cells were maintained at 1  ×  10⁵ cells/ml at 37°C and 5% CO₂ and periodically tested for expression of KIT and FcεRI by flow cytometry. The cells were used within 8 weeks of thawing from cryopreservation.

The LAD2 cell line (a gift from Arnold Kirshenbaum) was cultured in StemPro-34 SFM media (Life Technologies) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (PeproTech, Rocky Hill, NJ). Cells were maintained at 1 × 10⁵ cells/ml at 37 °C and 5% CO₂ and periodically tested for expression of c-KIT and FcεRI by flow cytometry.