Histopaque 1077 hybri maxtm

Manufactured by Merck Group

Sourced in Germany

Histopaque®-1077 Hybri-MaxTM is a sterile, endotoxin-tested, and cell culture tested solution designed for the separation of mononuclear cells from human peripheral blood or bone marrow. It is a density gradient medium that can be used to isolate mononuclear cells from whole blood or bone marrow samples.

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Lab products found in correlation

Sepmatetm tube, by STEMCELL (1 mentions) Sepmate tube, by STEMCELL (1 mentions) Pancoll, by PAN Biotech (1 mentions) Transit siquest reagent, by Mirus Bio (1 mentions) Bovine ifn γ elisa kit, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Rpmi 1640, by Lonza (1 mentions)

5 protocols using histopaque 1077 hybri maxtm

Peripheral Blood Mononuclear Cell Isolation

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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using Histopaque®-1077 Hybri-Max^TM (Sigma-Aldrich) density gradient centrifugation in SepMate^TM tubes (Stemcell) as previously described (20, 23, 24). Isolated PBMCs were cryopreserved in fetal calf serum containing 10% DMSO and stored in liquid nitrogen.

Reynolds C.J., Pade C., Gibbons J.M., Otter A.D., Lin K.M., Muñoz Sandoval D., Pieper F.P., Butler D.K., Liu S., Joy G., Forooghi N., Treibel T.A., Manisty C., Moon J.C., Semper A., Brooks T., McKnight Á., Altmann D.M., Boyton R.J., Abbass H., Abiodun A., Alfarih M., Alldis Z., Altmann D.M., Amin O.E., Andiapen M., Artico J., Augusto J.B., Baca G.L., Bailey S.N., Bhuva A.N., Boulter A., Bowles R., Boyton R.J., Bracken O.V., O’Brien B., Brooks T., Bullock N., Butler D.K., Captur G., Carr O., Champion N., Chan C., Chandran A., Coleman T., Couto de Sousa J., Couto-Parada X., Cross E., Cutino-Moguel T., D’Arcangelo S., Davies R.H., Douglas B., Di Genova C., Dieobi-Anene K., Diniz M.O., Ellis A., Feehan K., Finlay M., Fontana M., Forooghi N., Francis S., Gibbons J.M., Gillespie D., Gilroy D., Hamblin M., Harker G., Hemingway G., Hewson J., Heywood W., Hickling L.M., Hicks B., Hingorani A.D., Howes L., Itua I., Jardim V., Lee W.Y., Jensen M., Jones J., Jones M., Joy G., Kapil V., Kelly C., Kurdi H., Lambourne J., Lin K.M., Liu S., Lloyd A., Louth S., Maini M.K., Mandadapu V., Manisty C., McKnight Á., Menacho K., Mfuko C., Mills K., Millward S., Mitchelmore O., Moon C., Moon J., Muñoz Sandoval D., Murray S.M., Noursadeghi M., Otter A., Pade C., Palma S., Parker R., Patel K., Pawarova M., Petersen S.E., Piniera B., Pieper F.P., Rannigan L., Rapala A., Reynolds C.J., Richards A., Robathan M., Rosenheim J., Rowe C., Royds M., Sackville West J., Sambile G., Schmidt N.M., Selman H., Semper A., Seraphim A., Simion M., Smit A., Sugimoto M., Swadling L., Taylor S., Temperton N., Thomas S., Thornton G.D., Treibel T.A., Tucker A., Varghese A., Veerapen J., Vijayakumar M., Warner T., Welch S., White H., Wodehouse T., Wynne L., Zahedi D., Chain B, & Moon J.C. (2022). Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure. Science (New York, N.y.).

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Isolation and Cryopreservation of PBMCs

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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples using Pancoll (Pan Biotech) or Histopaque®-1077 Hybri-Max^TM (Sigma-Aldrich) density gradient centrifugation in SepMate tubes (StemCell) according to the manufacturer’s specifications. Isolated PBMCs were cryopreserved in fetal calf serum containing 10% DMSO and stored in liquid nitrogen.

Reynolds C.J., Swadling L., Gibbons J.M., Pade C., Jensen M.P., Diniz M.O., Schmidt N.M., Butler D.K., Amin O.E., Bailey S.N., Murray S.M., Pieper F.P., Taylor S., Jones J., Jones M., Lee W.Y., Rosenheim J., Chandran A., Joy G., Di Genova C., Temperton N., Lambourne J., Cutino-Moguel T., Andiapen M., Fontana M., Smit A., Semper A., O’Brien B., Chain B., Brooks T., Manisty C., Treibel T., Moon J.C., Noursadeghi M., Altmann D.M., Maini M.K., McKnight Á, & Boyton R.J. (2020). Discordant neutralizing antibody and T cell responses in asymptomatic and mild SARS-CoV-2 infection. Science Immunology, 5(54), eabf3698.

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Modulating Bovine Immune Response to LSDV

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Blood was collected from cattle at 6 months post-vaccination of a live-attenuated lumpy skin disease vaccine (Lumpi-ProVac^Ind). The PBMCs were isolated by using Histopaque®-1077 Hybri-Max^TM (Sigma, Steinheim, Germany) density gradient centrifugation. Cells were seeded into 6-well plates. The miR-29a inhibitor (100 nM final concentration) or negative control (Integrated DNA Technologies, Inc.) were transfected using the TransIT-siQUEST reagent (Mirus Bio, Madison, USA) according to the manufacturer’s instructions. After incubation for 24 h, cells were washed with PBS and stimulated with UV-inactivated LSDV antigen (2 µg/ml). The levels of IFN-γ released in the supernatant at 36 h post-stimulation were measured by the Bovine IFN-γ-ELISA kit (Invitrogen, Frederick, USA) according to the manufacturer’s instructions.

Kumar R., Kamboj H., Dhanda S., Verma A., Chander Y., Nehra K., Bhati A., Dedar R.K., Sharma D.K., Barua S., Tripathi B.N., Sharma S, & Kumar N. (2024). Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle. Virulence, 15(1), 2324711.

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UV-inactivated LSDV Antigen Stimulation of PBMCs

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The PBMCs were isolated by using Histopaque®-1077 Hybri-Max^TM (Sigma, Steinheim, Germany) density gradient centrifugation according to the instructions of manufacturer and resuspended in RPMI 1640 (Sigma, St Louis, USA) supplemented with 10% heat-inactivated foetal calf serum. Cell density was adjusted to 5 × 10⁶ cells/well in a 96 well plate. Live LSDV has toxic effect on PBMCs [42 ]. Therefore, for stimulation, PBMCs were treated with UV-inactivated-LSDV antigen (2 µg/ml) at 37°C in 5% CO₂ for 36 h.

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Primary Lamb and Vero Cell Culture

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Primary lamb testicle cells [40 (link)] and African green monkey kidney (Vero) cells [41 (link)] were available at the National Center for Veterinary Type Cultures (NCVTC), Hisar, and were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with antibiotics and 10–15% foetal calf serum. Peripheral blood mononuclear cells were separated from the cattle blood using Histopaque®-1077 Hybri-Max^TM (Sigma, Steinheim, Germany) and cultured in RPMI 1640 (Lonza, Walkersville, MD, USA) supplemented with antibiotics and 10% foetal calf serum.

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