Histopaque 1077 1119 gradient

Manufactured by Merck Group

Histopaque 1077/1119 gradient is a density gradient medium used for the isolation of mononuclear cells from whole blood. It is a sterile, endotoxin-tested solution composed of polysucrose and sodium diatrizoate adjusted to a density of 1.077 g/mL and 1.119 g/mL, respectively.

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Lab products found in correlation

Facscalibur, by BD (2 mentions) Cd16b, by BioLegend (2 mentions) Heparin coated vacutainers, by BD (2 mentions) Cd16b staining, by BioLegend (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Trypan blue, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Histopaque-1077 (1979 citations) Histopaque (690 citations) Histopaque 1119 (267 citations) Histopaque gradient (60 citations) Histopaque-1077 gradient (33 citations) Histopaque 1077/1119 (14 citations) Histopaque 1077 and 1119 gradients (2 citations)

5 protocols using histopaque 1077 1119 gradient

Isolation of Human Neutrophils

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Human neutrophils were isolated as previously described (58 (link)). Briefly, peripheral blood was collected from healthy, nonpregnant women and used immediately. Neutrophils were isolated by density centrifugation over a Histopaque 1077/1119 gradient (Sigma-Aldrich). Neutrophils were washed with Hank balanced salt solution without calcium or magnesium (Life Technologies), and hypotonic red blood cell (RBC) lysis was performed. The isolated neutrophil fraction was >95% pure, as determined by CD16b staining (1:100 dilution, no. 302005; BioLegend, San Diego, CA) by flow cytometric analysis (FACS Calibur; BD Biosciences). Human tissue collection was approved by the Yale University’s Human Research Protection Program (no. 0607001625).

Ren C., Yuan Q., Jian X., Randazzo P.A., Tang W, & Wu D. (2020). Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization. The Journal of Immunology Author Choice, 204(4), 1012-1021.

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Isolation of Peripheral Neutrophils

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Neutrophils were isolated from the peripheral blood of healthy, non-pregnant women and used immediately. Briefly, blood was collected by venepuncture into heparin-coated vacutainers (BD Biosciences, San Jose, CA) and red blood cells were sedimented using dextran (Sigma-Aldrich). Neutrophils were isolated by density centrifugation over a Histopaque 1077/1119 gradient (Sigma-Aldrich). Neutrophils were washed with Hank’s balanced salt solution without calcium or magnesium (Life Technologies) and hypotonic red blood cell lysis was performed. The isolated neutrophil fraction was >95% pure as determined by CD16b staining (#302005, Biolegend, San Diego, CA, 1:100 dilution) by flow cytometric analysis (FACs Calibur, BD Biosciences).

Tong M., Potter J.A., Mor G, & Abrahams V.M. (2019). LPS-Stimulated Human Fetal Membranes Induce Neutrophil Activation and Release of Vital Neutrophil Extracellular Traps. Journal of immunology (Baltimore, Md. : 1950), 203(2), 500-510.

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Isolation of Monocytes and Neutrophils from Bone Marrow

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All bone marrow cells were obtained, and the suspensions were filtered through 70 μm nylon mesh. The cells were carefully layered onto Histopaque-1077/1119 gradient solutions (Sigma) and were centrifuged at 700 ×g for 30 min. After centrifugation, the monocytes were aspirated from the “mononuclear” layer, and neutrophils were found in the “granulocyte” layer. Cells were collected and washed twice with sterile PBS. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin, streptomycin, HEPES and glutamine.

Liu L., Liu Y., Xu B., Liu C., Jia Y., Liu T., Fang C., Wang W., Ren J., He Z., Men K., Liang X., Luo M., Shao B., Mao Y., Xiao H., Qian Z., Geng J., Dong B., Mi P., Jiang Y., Wei Y, & Wei X. (2018). Negative regulation of cationic nanoparticle-induced inflammatory toxicity through the increased production of prostaglandin E2 via mitochondrial DNA-activated Ly6C+ monocytes. Theranostics, 8(11), 3138-3152.

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Isolation of Neutrophils from Healthy Women

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Neutrophils were isolated from the peripheral blood of healthy non-pregnant women and used immediately as previously described (Tong et al. 2019 ). Briefly, blood was collected into heparin-coated vacutainers (BD Biosciences, San Jose, CA) and neutrophils were isolated by density centrifugation on a Histopaque 1077/1119 gradient (Sigma-Aldrich). Hypotonic red blood cell lysis was performed and the isolated neutrophil fraction was >95% pure as determined by CD16b staining (#302005, Biolegend, San Diego, CA, 1:100 dilution).

Tong M., Hanna S.E, & Abrahams V.M. (2021). Polymicrobial Stimulation of Human Fetal Membranes Induce Neutrophil Activation and Neutrophil Extracellular Trap Release. Journal of reproductive immunology, 145, 103306.

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Isolation and Characterization of Neutrophils

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Isolation of neutrophils was performed as described by Siemsen et al. (2007) (link). In short, plasma was removed after centrifugation and red blood cells were lysed by adding 50 mL of endotoxin-free purified water. Five milliliters of NaCl (10%; Sigma-Aldrich, Bornem, Belgium) was added and samples were centrifuged at 740 × g for 10 min at room temperature with low braking speed. The resulting leukocyte suspension was layered carefully on top of a Histopaque 1077/1119 gradient (Sigma-Aldrich). The final pellet was resuspended in 1 mL of PBS (Sigma-Aldrich). Cell viability was determined in triplicate (3 × 100 cells were counted) by the Trypan blue (Invitrogen, Merelbeke, Belgium) exclusion test using a Bürker chamber. Neutrophil purity was analyzed by bright-field microscopy with a 100× objective and immersion oil (Axioskop 2 MOT, Carl Zeiss N.V., Oberkochen, Germany) by staining cells with Sangodiff G (Sigma-Aldrich). Purity was also determined in triplicate (3 × 100 cells were counted).

Boulougouris X., Rogiers C., Van Poucke M., De Spiegeleer B., Peelman L.J., Duchateau L, & Burvenich C. (2019). Distinct neutrophil C5a receptor inflammatory events in cows initiated by chemoattractant C5a and lipopolysaccharide around parturition and in mid lactation. Journal of dairy science, 102(2).

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