Cyclophosphamide hydrate is a DNA alkylating agent, an inhibitor of DNA synthesis. Cyclophosphamide hydrate has antitumor and immunosuppressive activities.

方法：巨噬细胞 Raw 264.7 用 Cyclophosphamide hydrate (10-250 μg/mL) 处理 48 h，使用 MTT assay 检测细胞活力。
结果：Cyclophosphamide hydrate 对 Raw 264.7 有细胞毒性，IC50 为 145.44 μg/mL。[1]
方法：人乳腺癌细胞 MDA-MB-231 和 MDA-MB-435S 用 Cyclophosphamide hydrate (0.25-1 mM) 处理 4-24 h，使用 Wound healing assay 检测细胞迁移情况。
结果：迁移的 MDA-MB-231 细胞数量的增加取决于 Cyclophosphamide 的浓度，而到 MDA-MB-435S 细胞的迁移显著减少，并且与 Cyclophosphamide 浓度无关。[2]

方法：为检测体内抗肿瘤活性，将 Cyclophosphamide hydrate (50-150 mg/kg) 单次腹腔注射给携带小鼠结直肠肿瘤 CT26 的 BALB/c 小鼠。
结果：Cyclophosphamide hydrate 诱导肿瘤体积显著减少。[3]
方法：为检测体内抗肿瘤活性，将 Cyclophosphamide hydrate (140 mg/kg) 口服给药给携带小鼠乳腺癌肿瘤 4T1 的 Balb/c 小鼠，每六天一次，持续十八天。
结果：Cyclophosphamide hydrate 治疗显著抑制了小鼠的肿瘤生长。[4]

9L cells are treated with drug for the times indicated in each experiment. Floating and attached cells are collected, pooled, resuspended in lysis buffer (10 mM HEPES buffer, pH 7.4, containing 2 mM EDTA, 0.1% CHAPS detergent, 5 mM DTT, 350 ng/mL phenylmethylsulfonyl fluoride, 10 ng/mL pepstatin A, 10 ng/mL aprotinin, and 20 ng/mL leupeptin) and lysed by three freeze-thaw cycles (alternating between a dry ice isopropanol bath and a 37°C water bath). Lysates are spun in a bench top centrifuge at full speed for 15 min and the supernatant (cell extract) fraction transferred to a new tube. Cell extracts (20 μL) are assayed for caspase 9, caspase 8, and caspase 3 activity by incubation at 37°C for either 1 h (caspase 3) or 3 h (caspase 9 and caspase 8) in 500 μL of reaction buffer (10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, and 5 mM DTT) containing 50 μM caspase form-selective substrate: Ac-LETD-AFC for caspase 8; Ac-LEHD-AFC for caspase 9; and Ac-DEVD-AMC for caspase 3. Background activity is determined for each sample as follows. Cell extracts are preincubated for 15 min at room temperature, with or without caspase form-selective inhibitor: 1 μM z-LETD-FMK for caspase 8, 1 μM z-LEHD-FMK for caspase 9, and 5 μL of Casputin for caspase 3. Caspase activity measured in the absence of inhibitor is divided by the background caspase activity measured in the presence of inhibitor. A value of 1 is subtracted from each measured activity, such that a caspase activity of 0 corresponds to no increase in the specific caspase activity with drug treatment. Fluorescence of the caspase product (excitation at 395 nm and emission at 525 nm for AFC substrates, and excitation at 380 nm and emission at 460 nm for the AMC substrate) is measured using a Shimadzu model RF-1501 spectrofluorophotometer and the manufacturer's PC-1501 software package.

9L/pBabe, 9L/Bax, and 9L/Bcl-2 cells are treated with 12, 24, or 50 μM MFA for 72 h. Cells remaining on the plates at 0, 24, 48, and 72 h are washed twice with cold PBS and then stained for 5 min with crystal violet [1.25 g of crystal violet dissolved in a solution containing 50 mL of 37% formaldehyde and 450 mL of methanol]. The stained cells are washed three times in tap water and the plates are allowed to dry. The stain is eluted from the cells with 70% ethanol and the absorbance is then read at 595 nm. The staining intensity of each drug-treated sample (A?595) is then graphed as a percentage of the staining intensity at the 0-h time point.