Zymefree

Manufactured by HiMedia

ZymeFree™ is a laboratory equipment designed for enzymatic reactions. It provides a controlled environment for the performance of various biochemical assays and experiments.

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Lab products found in correlation

Fetal bovine serum (fbs), by PAN Biotech (2 mentions) Gentamycin, by Merck Group (1 mentions) Tib 71, by American Type Culture Collection (1 mentions) Higlutaxl rpmi 1640, by HiMedia (1 mentions) Streptomycin, by HiMedia (1 mentions) Accutase, by HiMedia (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Pharm lyse solution, by BD (1 mentions) L glutamine, by HiMedia (1 mentions) Rpmi 1640, by PAN Biotech (1 mentions)

4 protocols using zymefree

Chikungunya Virus Infection in Murine Macrophages

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DRDE-06 (accession no. EF210157.2), an Indian outbreak strain of CHIKV and Vero cells (African green monkey kidney epithelial cell line) were kind gifts from Dr. M. M. Parida, DRDE, Gwalior, India. The mouse monocyte/macrophage cell line, Raw264.7 (ATCC® TIB-71™) was maintained in RPMI-1640 (HiGlutaXL™ RPMI-1640) supplemented with 2.0 mM L-glutamine, Penicillin 100 U/ml, Streptomycin 0.1 mg/ml (Himedia Laboratories Pvt. Ltd, MH, India), 10% Fetal bovine serum (FBS; PAN Biotech, Germany) at 37°C under a humidified incubator with 5% CO₂. The Vero cells were maintained in Dulbecco's modified Eagle's medium (DMEM; PAN Biotech, Germany) supplemented with 5% FBS, Gentamycin (Sigma-Aldrich, MO, USA). The enzyme-free cell dissociation reagent (ZymeFree™; Himedia Laboratories Pvt. Ltd, MH, India) was used for the maintenance of the Raw264.7 cells.
Eight to ten weeks old male or female BALB/c mice were used for this experiment. The animals used in these experiments were approved by Institutional Animal Ethics Committee, NISER and followed the guidelines by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Nayak T.K., Mamidi P., Sahoo S.S., Kumar P.S., Mahish C., Chatterjee S., Subudhi B.B., Chattopadhyay S, & Chattopadhyay S. (2019). P38 and JNK Mitogen-Activated Protein Kinases Interact With Chikungunya Virus Non-structural Protein-2 and Regulate TNF Induction During Viral Infection in Macrophages. Frontiers in Immunology, 10, 786.

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Establishment of Glioblastoma Cell Cultures

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The Human Glioblastoma Cell line LN-18 was obtained from American Type Culture Collection (ATCC Rockville, USA). The Human Glioblastoma tumor tissue samples were collected from surgeries performed at Sasoon hospital, DY Patil Hospital and Inamdar hospital, Pune. Informed consent was obtained from patients for tissue procurement in accordance with the protocol approved by the institutional ethics committee of NCCS and graded by pathologist. Primary Cultures were obtained by processing GBM tumor samples using Accutase (Himedia) and Zymefree (Himedia) to obtain adherent cell cultures which were passaged to 3–10 passages. We have previously reported the expression of neuronal markers in primary cultures-G162 (link). Cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, supplemented with 5% heat inactivated fetal calf serum (Gibco) in a humidified incubator at 37 °C with 5% CO₂. Cells were dislodged using trypsin (0.125%) – EDTA (0.02%) solution.

Chandrika G., Natesh K., Ranade D., Chugh A, & Shastry P. (2016). Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling. Scientific Reports, 6, 22455.

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Isolation of Murine Lung Macrophages

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Mice were euthanized by using a Ketamine–Xylazine cocktail (2 mg ketamine + 0.2 mg xylazine/20 g of mice). Lung tissue was collected and chopped with a fine blade in RPMI. The chopped tissues were transferred to digestion enzyme cocktail of hyaluronidase (200 μg/ml), collagenase-A (700 μg/ml), and DNase (30 μg/ml) and incubated at 37°C for 45 min in shaking condition. To get a single-cell suspension, the extracellular matrix was removed by passing the digested lung tissue through a cell strainer (pore size 40 μm). RBCs were lysed using BD Pharm lyse solution. After RBC lysis, cells were allowed to adhere in T75 culture flask for 5 h; non-adherent cells were removed by washing twice with PBS. Culture flasks were maintained on ice throughout washing and tapped firmly in between. After washing, strongly adherent cells (mainly macrophages) were removed by using Zymefree (non-enzymatic cell dissociation solution from Himedia). Purity of these cells were determined by flow cytometry. About 80% of cells were CD11b⁺, CD14⁺, and F4/80⁺ (Figure S6).

Pal L., Nandani R., Kumar P., Swami B., Roy G, & Bhaskar S. (2021). Macrophages Are the Key Players in Promoting Hyper-Inflammatory Response in a Mouse Model of TB-IRIS. Frontiers in Immunology, 12, 775177.

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Culturing Mouse Macrophage and Human Monocyte Cells

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Mouse macrophage cell line, RAW 264.7 (source – ATCC (ATCC® TIB-71™)) was cultured in complete Roswell Park Memorial Institute-1640 medium (RPMI-1640) (PAN Biotech, Aidenbach, Germany) with penicillin (100 U/mL), Streptomycin (0.1 mg/mL), and 2.0 mM L-Glutamine (Himedia Laboratories Pvt. Ltd., Mumbai, MH, India), 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech, Aidenbach, Germany) at 37ºC in a sterile incubator with 5% CO₂ and appropriate humidity. Enzyme-free cell dissociation reagent (ZymeFree™; Himedia Laboratories Pvt. Ltd, Mumbai, MH, India) was used to maintain the cells [56 ].
Undifferentiated human leukemia monocytic cell line, THP-1 (source – ATCC (ATCC® TIB-202™)) was maintained in complete RPMI-1640 (PAN Biotech, Aidenbach, Germany) supplemented with Penicillin (100 U/mL), Streptomycin (0.1 mg/mL), and 2.0 mM L-Glutamine (Himedia Laboratories Pvt. Ltd., Mumbai, MH, India), 10% heat-inactivated FBS (PAN Biotech, Aidenbach, Germany) at 37ºC in a sterile incubator with 5% CO₂ and appropriate humidity. THP-1 cells were further treated with 100 ng/ml PMA for 24 h to differentiate monocytes into macrophage-like cells [75 ].

Radhakrishnan A., Mukherjee T., Mahish C., Kumar P.S., Goswami C, & Chattopadhyay S. (2023). TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro. BMC Immunology, 24, 16.

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