Freezing media

Manufactured by Thermo Fisher Scientific

Freezing media is a liquid solution designed to protect cells and tissues during the freezing process. It helps to maintain cell viability and integrity when samples are cryogenically stored at ultra-low temperatures.

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Recovery Cell Culture Freezing Media (10 citations) Recovery freezing media (2 citations) OCT freezing media (2 citations) Recovery Cell Freezing Media (2 citations)

4 protocols using freezing media

Dissociation and Cryopreservation of HCC Tissues

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HCC tumor tissues and adjacent tissues were collected after surgical resection in MACS Tissue Storage Solution (Miltenyi Biotec) in a 50 mL conical tube and transported on ice to the laboratory. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm³) on ice, and enzymatically digested with collagenase I (Worthington) for 15 min at 37 °C, with agitation. After digestion, samples were sieved through a 70 µm cell strainer, and centrifuged at 300 g for 5 min. The cell pellet was resuspended in 1 mL freezing media (Gibco) for long-term cryopreservation in liquid nitrogen. Throughout the dissociation procedure, cells were maintained on ice whenever possible, and the entire procedure was completed in less than 1 hr.

Meng Y., Sang Y., Liao J., Zhao Q., Qu S., Li R., Jiang J., Wang M., Wang J., Wu D., Cheng C, & Wei L. (2022). Single cell transcriptional diversity and intercellular crosstalk of human liver cancer. Cell Death & Disease, 13(3), 261.

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Fibroblast Cell Culture Protocols

Cited 1 time

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Fibroblast cell lines were obtained from the Coriell Institute for Medical Research (Camden, NJ) from four male humans and chimpanzees (Pan troglodytes; supplementary table S1, Supplementary Material online). Our cell culture methods approximately followed those of Chang et al. (2004) (link) and Iyer et al. (1999) (link). Briefly, we seeded cells in media with FBS (Hyclone defined FBS (-)HI, Fisher) at ∼50% confluency and grew overnight. At 60% confluency one set of plates were set aside and processed as described below (fig. 1A, “Pre-challenge” time point). The remainder of the cells were then incubated in starvation media (0.1% FBS) for 48 h, after which the growth media was replaced. Collections were then done at time 0, 12 h, and 24 h. All cells for the RNA-Seq and DNase-Seq experiments were from the same batch and collected at the same time. For the RNA-Seq assays, cells were rinsed with Qiazol (Qiagen) and vortexed. The RNA was isolated using an RNeasy kit (Qiagen), with a DNaseI treatment. For the DNase-Seq assays, ∼20 million cells were spun down and slowly frozen in freezing media (Gibco) to −80 °C.

Pizzollo J., Nielsen W.J., Shibata Y., Safi A., Crawford G.E., Wray G.A, & Babbitt C.C. (2018). Comparative Serum Challenges Show Divergent Patterns of Gene Expression and Open Chromatin in Human and Chimpanzee. Genome Biology and Evolution, 10(3), 826-839.

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Tumor Tissue Dissociation and Cryopreservation

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Core needle or resected tumor biopsies were collected in RPMI 1640 media supplemented with penicillin/streptomycin and immediately transferred for dissociation within minimum ischemic time. On arrival, each sample was placed in a sterile Petri dish on ice. Fat, fibrous and necrotic areas were removed, and the tissue was subsequently minced to smaller pieces of less than 3 mm. Sample pieces were transferred to a gentleMACS C Tube (Miltenyi #130-093-237) containing 5 ml digestion enzyme mix previously prepared according to the kit user guide (Tumor dissociation Kit from Miltenyi Biotech # 130-095-929). The C tube was then placed on a gentleMACS™ Octo Dissociator for mechanical dissociation using program h_tumor_01. Upon run completion, the sample was incubated for 30 min at 37 °C under continuous low shaking. Subsequently, the sample was filtered using a 70-gm nylon mesh (Miltenyi # 130-095-823). 10 μl of this cell suspension was counted by Trypan Blue to determine the concentration of live cells. The sample was then centrifuged at 300×g and 4 °C for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 1 ml freezing media (Gibco) for long term cryopreservation in liquid nitrogen. Throughout the dissociation procedure, cells were maintained on ice whenever possible, and the entire procedure was completed in less than 1 hr.

Ma L., Hernandez M.O., Zhao Y., Mehta M., Tran B., Kelly M., Rae Z., Hernandez J.M., Davis J.L., Martin S.P., Kleiner D.E., Hewitt S.M., Ylaya K., Wood B.J., Greten T.F, & Wang X.W. (2019). Tumor cell biodiversity drives microenvironmental reprogramming in liver cancer. Cancer cell, 36(4), 418-430.e6.

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Isolation and Cultivation of Breast Fibroblasts

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Breast fibroblast cells were obtained and used as previously described [21 (link)]. NBFs (normal breast fibroblasts) are cultures obtained from tissues derived from reduction mammoplasty, CAFs (cancer-associated fibroblasts), and TCFs (tumor counterpart fibroblasts) are from histologically normal parts of tumors. Fibroblasts were cultured in M199 medium (Gibco, Grand Island, NY, USA ) and Ham’s F12 (Gibco) mixed 1:1 and supplemented with 20% Heat Inactivated Fetal Bovine Serum (FBS) (Gibco) and 1% Antibiotic-Antimycotic 100X (Gibco). Breast cancer cells MDA-MB-231 and MCF-7 were obtained from ATCC and were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 1% Antibiotic-Antimycotic, while Human Mammary Luminal Epithelial (HMLE) cells were a generous gift from Dr. Hazem Ghebeh (KFSHRC) and were maintained in DMEM/F12 (Gibco) supplemented with 0.5% Bovine Pituitary Extract (Gibco) and 1% HuMEC supplement (Gibco) in addition to 2% FBS and 1% Antibiotic-Antimycotic. Cells were maintained at 37°C in humidified incubator with 5% CO₂. For long storage, cells were kept in freezing media (Gibco). Recombinant IL-6 was purchased from GenWay Biotech. Recombinant OPG was purchased from Sigma-Aldrich.

Al-Nasrallah H.K., Al-Ansari M.M, & Aboussekhra A. (2022). Osteoprotegerin (OPG) Upregulation Activates Breast Stromal Fibroblasts and Enhances Their Pro-Carcinogenic Effects through the STAT3/IL-6 Signaling. Cells, 11(21), 3369.

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