Atcc crl 2539

The murine mammary carcinoma 4T1 cells (ATCC^® CRL-2539™) were obtained from ATCC (Rockville, MD, USA). The cells were grown in RPMI-1640 with 100 units/mL penicillin, 100 µg/mL streptomycin, and 12.5 units/mL nystatin (Biological Industries, Beit Haemek, Israel), and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Cell viability assessment by the Hoechst assay was performed as described [42 (link)].

MDA-MB-231(ATCC® HTB-26™) human breast tumor cell line and 4T1 (ATCC® CRL-2539™) mouse breast tumor cell line were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Chinese Hamster Ovary (CHO-K1)(Cell bank of Chinese Academy of Science, Beijing, China) . MDA-MB-231 cells and 4T1 cells were grown in DMEM(gibco, life technologies, China) medium or RPMI-1640 (gibco, life technologies, China)medium respectively supplemented with 10 % fetal bovine serum (FBS, gibco, life technologies, China),and penicillin(100units/mL) and streptomycin(100units/mL) incubated at 37 °C in a 5 % CO₂ –95 % air environment. CHO cells were cultured in the same condition as MDA-MB-231 cells.

The 4T1 mouse mammary tumor cell line was purchased (ATCC #CRL-2539, American Type Culture Collection, Manassas, VA, USA) within the last 12 months and passaged less than five times before implantation into the mice. Absence of rodent pathogen infection of the 4T1 cells including mycoplasma was confirmed with a polymerase chain reaction (IDEXX BioAnalytics, Columbia, MO, USA). The mice were anesthetized (isoflurane, 1–3%), and 4T1 cells (1 × 10⁶ cells in 100 µL phosphate-buffered saline (PBS) per mouse) were inoculated unilaterally in the third inguinal mammary fat pad. The next day, the mice were inoculated with intraperitoneal sterile saline (0.9% sodium chloride injection, USP) or pharmaceutical grade Dox (6 or 10 mg/kg) (Athenex, Buffalo, NY, USA). As Dox was supplied in a saline solution (2 mg/mL), the injection volume was adjusted according to the dose received and did not exceed 200 µL. The sterile saline or Dox injection was repeated after 1 week (total, two injections per mouse). The tumor length (L) and width (W) were measured twice weekly with a digital caliper and were calculated with the equation: V = L × (W²)/2.

4T1—a highly aggressive and metastatic triple-negative breast cancer cell line; A549—less aggressive non-small-cell lung carcinoma cells; B16F10—melanoma cells, frequently utilized in metastasis research; CaCo-2—a heterogenous culture of colon adenocarcinoma cells; MDA-MB-231—an aggressive type of triple-negative breast cancer cells, often used to model late-stage breast cancer; SK-BR-3—a HER2-overexpressing breast cancer cell line; and RAW 264.7—a macrophage-like cell line, established from a leukemia-induced mouse tumor, were obtained from the American Type Culture Collection as ATCC CRL-2539, ATCC CRM-CCL-185, ATCC CCL-185, ATCC CRL-6475, ATCC CRM-HTB-26, ATCC HTB-30, and ATCC SC-6003, respectively. RAW 264.7 are often used as a macrophage model; they respond to LPS stimulation and are able to produce nitric oxide. Primary tumor cells from a transgenic PyMT mouse breast cancer model, which closely mimics human breast cancer progression, were isolated and grown as described previously [33 (link)]. Cells were grown in DMEM (Sigma) medium containing 10% FBS, 1% P/S, and 1% Glutamax.