Fix and perm medium a

Manufactured by Thermo Fisher Scientific

Sourced in United States

Fix and Perm Medium A is a product designed for use in laboratory settings. It serves as a fixative and permeabilization medium, facilitating the preparation of samples for downstream analysis. The core function of this product is to preserve and permeabilize cellular structures, enabling effective processing and examination of samples.

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Lab products found in correlation

4 protocols using fix and perm medium a

Cell Viability and Apoptosis Assays

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500,000 cells were plated in wells of a 24-well plate, and treated with DMSO or USP7i as indicated in the Figure Legends. Cells were counted each day via trypan blue, and inhibitor/medium was changed. When cells reached a confluency of >1 million cells/ml, cells were diluted 1:2, and this dilution was factored into the cell numbers for viability assays. For apoptosis analysis, cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Life Technologies) according to the manufacturer’s protocol, except that cells were stained for 20 min at 4°C, prior to staining with PE-conjugated Annexin V (Life Technologies) in Annexin V Binding Buffer (BD Biosciences, San Jose, CA), according to the manufacturers’ instructions. To measure cell cycle, cells were fixed in 100 μl Fix and Perm Medium A (Life Technologies) for 15 min, washed with PBS, and incubated with 100 μl Fix and Perm Medium B (Life Technologies) + 1 μg/ml DAPI (Invitrogen, Carlsbad, CA) for 1h at 4°C. Flow cytometry was performed on an LSR II (BD, Franklin Lakes, NJ) and analyses were performed using FlowJo software (Tree Star, Ashland, OR). Statistical analyses were performed using GraphPad Prism software (GraphPad Software). Comparisons were made using the Student’s unpaired, two-sided t-test. p values <0.05 were considered statistically significant.

Jin Q., Martinez C.A., Arcipowski K.M., Zhu Y., Diaz B.T., Wang K.K., Johnson M.R., Volk A.G., Wang F., Wu J., Grove C., Wang H., Sokirniy I., Thomas P.M., Goo Y.A., Abshiru N.A., Hijiya N., Peirs S., Vandamme N., Berx G., Goosens S., Marshall S.A., Rendleman E.J., Takahashi Y.H., Wang L., Rawat R., Bartom E.T., Collings C.K., Van Vlierberghe P., Strikoudis A., Kelly S., Ueberheide B., Mantis C., Kandela I., Bourquin J.P., Bornhauser B., Serafin V., Bresolin S., Paganin M., Accordi B., Basso G., Kelleher N.L., Weinstock J., Kumar S., Crispino J.D., Shilatifard A, & Ntziachristos P. (2018). USP7 cooperates with NOTCH1 to drive the oncogenic transcriptional program in T cell leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(1), 222-239.

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Isolation and Culture of Human Endothelial Cells

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Ham’s F12, Medium 199, Human Endothelial-SFM, fetal bovine serum (FBS), Dulbecco’s Phosphate-Buffered Saline (PBS), Insulin/Transferrin/Selenium (ITS), Collagenase Type I, TrypLE^TM Express (TE), TrypLE^TM Select (TS), gentamicin, amphotericin B, 5-ethynyl-29-deoxyuridine (EdU) incorporation Click-iT Alexa Fluor 488 cell proliferation assay kit, Fix and Perm (Medium A), penicillin and streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), trypan blue (0.4%), alizarin red, paraformaldehyde (PFA), human serum (HS), Collagen IV from human placenta, ascorbic acid, and ascorbate-2-phosphate were purchased from Sigma (St. Louis, MO, USA). Recombinant basic fibroblast growth factor was bought from R&D Systems (bFGF, Minneapolis, MN, USA). Rho-associated, coiled-coil protein kinase inhibitor Y-27632 was brought from Miltenyi Biotec (Bergisch Gladbach, Germany). FNC coating mixture was obtained from United States Biologicals (Swampscott, MA, USA). Liberase TH was purchased from Roche (Mannhein, Germany). EquaFetal® was from Atlas Biologicals (Fort Collins, CO, USA). FREEZEstem cryo-preservation medium, LAMSCREEN^TM, human recombinant laminin-511 and 521 were bought from BioLamina (Sundbyberg, Sweden). Finally, Cryoscarless cryo-preservation medium was from Funakoshi (Tokyo, Japan).

Peh G.S., Ang H.P., Lwin C.N., Adnan K., George B.L., Seah X.Y., Lin S.J., Bhogal M., Liu Y.C., Tan D.T, & Mehta J.S. (2017). Regulatory Compliant Tissue-Engineered Human Corneal Endothelial Grafts Restore Corneal Function of Rabbits with Bullous Keratopathy. Scientific Reports, 7, 14149.

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Multiparametric Analysis of CNS-Derived Cells

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Cells isolated from the CNS were stimulated with PMA (500 ng/mL; Sigma Aldrich, St. Louis, MO), ionomycin (50 ng/mL; Sigma Aldrich, St. Louis, MO), and 1 μL/mL Golgiplug (BD Biosciences, Franklin Lakes, NJ) for 4 h at 37°C. Cells were washed with PBS containing 3% FBS. Surface antigens were stained with Abs in 100 μL of PBS/3% FBS for 20 min at 4°C. Cells were then fixed with 100 μL Fix and Perm Medium A (Thermo Fisher, Waltham, MA) for 20 min at room temperature and then permeabilized with Fix and Perm Medium B (Thermo Fisher, Waltham, MA) and stained with Abs against intracellular antigens in 100 μL Fix and Perm Medium B and 100 μL PBS/3%FBS for 1 h. Cells were then washed twice, resuspended in 500 μL PBS and analyzed on a BD FACSAria Fusion flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Hwang D., Boehm A., Rostami A., Zhang G.X, & Ciric B. (2021). Oral D-mannose treatment suppresses experimental autoimmune encephalomyelitis via induction of regulatory T cells. Journal of neuroimmunology, 362, 577778.

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Flow Cytometric Analysis of Stimulated Cells

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Isolated cells were stimulated with PMA (500 ng/mL; Sigma Aldrich), ionomycin (50 ng/mL; Sigma Aldrich), and 1 µL/mL Golgiplug (BD Biosciences) for 4 h at 37 o C. After stimulation, cells were washed with PBS containing 3% FBS (v/v). Cell surface antigens were stained with Abs in 100 µL of PBS/3% FBS for 20-30 min at 4 o C. Cells were then washed and fixed with 100 µL Fix and Perm Medium A (Thermo Fisher) for 20 min at room temperature and washed again. Cells were permeabilized with Fix and Perm Medium B (Thermo Fisher) and stained with Abs against intracellular antigens in 100 µL Fix and Perm Medium B and 100 µL PBS/3%FBS for 1 h. Cells were then washed twice, resuspended in 500 µL PBS and analyzed on a BD FACSAria Fusion flow cytometer (BD Biosciences).

Hwang D., Seyedsadr M., Ishikawa L.L.W., Boehm A., Sahin Z., Casella G., Jang S., Gonzalez M.V., Garifallou J., Hákonarson H., Zhang W., Xiao D., Rostami A., Zhang G., & Ćirić B. (2020). CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.

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