Ack lysis buffer

Manufactured by Quality Biological

Sourced in United States

ACK lysis buffer is a solution used to lyse red blood cells (erythrocytes) in order to isolate other cell types, such as leukocytes, from whole blood samples. The buffer contains ammonium chloride, potassium bicarbonate, and EDTA, which together effectively lyse red blood cells without damaging the remaining cells.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions) Dnase 1, by Merck Group (4 mentions) Facsaria 2, by BD (4 mentions) Fortessa x20, by BD (4 mentions) Hepes, by Thermo Fisher Scientific (4 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions) Collagenase d, by Merck Group (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Octo dissociator, by Miltenyi Biotec (3 mentions) Tumor dissociation kit, by Miltenyi Biotec (3 mentions) Dnase 1, by Roche (3 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (3 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Biomasher tube, by Nippi (2 mentions) Percoll, by GE Healthcare (2 mentions) Dimethyl sulfoxide (dmso), by Avantor (2 mentions) β mercaptoethanol, by Bio-Rad (2 mentions) Sepmate 15, by STEMCELL (2 mentions)

78 protocols using ack lysis buffer

Breast Tumor and Liver Metastasis Dissociation

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To obtain single-cell suspensions from breast tumors, tumors were harvested, weighed, diced, and then dissociated using a tumor dissociation kit (Miltenyi Biotec, cat. 130–096-730) and the OctoDissociator (Miltenyi Biotec) per the manufacturer’s instructions. The 37C_m_TDK_2 program was used to dissociate tumors per the manufacturer’s instructions. Samples were filtered using a 40 μm cell strainer and red blood cells were lysed using ACK lysis buffer (Quality Biological, cat. 118–156-721). The resulting single-cell suspensions were used for subsequent isolation of specific immune cell types or flow cytometry as described below. Blood from neu-N mice was obtained via retro-orbital bleeds as per protocol. Red blood cells were lysed using ACK lysis buffer (Quality Biological, cat. 118–156-721). Isolated cells were analyzed via flow cytometry.
To obtain single-cell suspensions of livers containing metastases in the PDAC model, livers were harvested and processed by mashing the liver through 100 μm and 40 μm cell strainers as previously described (35 ). Red blood cells were lysed using ACK lysis buffer (Quality Biological, cat. 118–156-721), and liver pellets were resuspended in 40% Percoll (GE Healthcare Life Sciences, cat. 17–0891-01) and underlayed with 80% Percoll. The immune cell layer was then removed and analyzed via flow cytometry.

Christmas B.J., Rafie C.I., Hopkins A.C., Scott B.A., Ma H.S., Cruz K.A., Woolman S., Armstrong T.D., Connolly R.M., Azad N.A., Jaffee E.M, & Roussos Torres E.T. (2018). Entinostat converts immune-resistant breast and pancreatic cancers into checkpoint-responsive tumors by reprogramming tumor-infiltrating MDSCs. Cancer immunology research, 6(12), 1561-1577.

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Isolation and Immunophenotyping of Mouse Bone Marrow Cells

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The bone marrow (BM) cells were flushed from the femurs or tibias, and the red blood cells were lysed using ACK lysis buffer (Quality Biological, Gaithersburg, MD). The cells were then stained for 20 min at 4 °C with conjugated anti-mouse antibodies (see Table 1 for a list of the antibodies that were used). After this time, cells were washed with PBS containing 1% FBS and analyzed by either Gallios or Cytoflex flow cytometers and Kaluza or CytExpert software (all from Beckman Coulter, Indianapolis, Indiana 46268, USA).

Awida Z., Bachar A., Saed H., Gorodov A., Ben-Califa N., Ibrahim M., Kolomansky A., Iden J.A., Graniewitz Visacovsky L., Liron T., Hiram-Bab S., Brines M., Gabet Y, & Neumann D. (2021). The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenesis In Vitro and Increases Bone Mineral Density in Mice. International Journal of Molecular Sciences, 23(1), 55.

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Isolation and Preparation of Immune Cells

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Heparin-treated blood was collected and lysed with ACK lysis buffer (Quality Biological) to isolate PBMCs. Lungs, liver, kidneys and tumors were collected in digestion media containing Roswell Park Memorial Institute (RPMI) 1640, 10% FCS, 50 U ml–1 DNase I (Sigma-Aldrich) and 0.2 mg/ml collagenase D (Sigma-Aldrich). Tissues were mechanically disrupted using the respective programs on the gentleMACS dissociator (Miltenyi Biotec) and incubated at 37 °C for 30–45 min in a shaking incubator. Spleens were mechanically disrupted and lysed with ACK lysis buffer. Lymph nodes were mechanically disrupted in BioMasher tubes (Nippi). All single-cell suspensions were filtered through a 70-μm cell strainer and resuspended in PBS for flow cytometry staining.

Baharom F., Ramirez-Valdez R.A., Khalilnezhad A., Khalilnezhad S., Dillon M., Hermans D., Fussell S., Tobin K.K., Dutertre C.A., Lynn G.M., Müller S., Ginhoux F., Ishizuka A.S, & Seder R.A. (2022). Systemic vaccination induces CD8+ T cells and remodels the tumor microenvironment. Cell, 185(23), 4317-4332.e15.

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Blood Collection and Processing for Vaccination Studies

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Blood was collected by venipuncture prior to vaccination, and on days 7, 10, 15, 30, 90 and 365. Approximately 64 mLs of blood were drawn into CPT tubes at each timepoint. Additionally, approximately 3 mLs of blood were drawn into serum tubes. Blood was centrifuged at 1600 rcf for 30 min. The serum fraction was removed and aliquoted for further studies. The cell layer was harvested and lysed with 5 mLs of ACK lysis buffer (Quality biological #118-156-101) for 5 minutes. After which cells were washed with PBS + 2% FBS three times after spinning at 1200 rpm for 8 minutes. Cells were then resuspended in the appropriate buffer for counting and further experiments.

Adekunle O., Dretler A., Kauffman R.C., Cho A., Rouphael N, & Wrammert J. (2021). Longitudinal analysis of human humoral responses after vaccination with a live attenuated V. cholerae vaccine. PLoS Neglected Tropical Diseases, 15(9), e0009743.

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Isolation of Mouse CD4+ T Cells

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Eight week-old mice were euthanized and single-cell suspensions were made of the spleen or thymuses in cold Hank’s balanced saline solution (HBSS) with 1% fetal bovine serum (FBS) (Invitrogen). After the removal of contaminating red blood cells using ACK lysis buffer (Quality Biological, Inc., Gaithersburg, MD), CD4+ T cells were isolated using the mouse CD3⁺ CD4+ T-cell enrichment column (R&D System, Minneapolis, MN) or using mouse CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and kept in RPMI supplemented with 10% FBS (Invitrogen) and 50 μM β-ME under 5% CO₂ at 37°C.

Lee J.H., Patel K., Tae H.J., Lustig A., Kim J.W., Mattson M.P, & Taub D.D. (2014). Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways. FEBS letters, 588(24), 4708-4719.

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Splenocyte IFNγ ELISpot Assay

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Spleens from mice were collected individually in RPMI 1640 media supplemented with 10% fetal bovine serum and penicillin, streptomycin, nonessential amino acids, sodium pyruvate, and β-mercaptoethanol (complete media) and then processed into single-cell suspensions and passed through a 70-μm nylon filter. Cell pellets were re-suspended in 3 ml of ACK lysis buffer (Quality Biological Inc., catalog no. 118156101) for 5 min on ice; then, PBS was added to stop the reaction. The samples were centrifuged at 400g for 5 min at 4°C, and cell pellets were re-suspended in complete media. ELISpot assays were performed using the Mouse IFNγ ELISpot Set (BD, catalog no. BD551083). Ninety-six–well ELISpot plates precoated with capture antibody overnight at 4°C were blocked with complete media for 2 hours at RT. Mouse splenocytes (500,000) were plated into each well and stimulated overnight with 1 μg per peptide per well of Spike-derived overlapping peptides. The spots were developed on the basis of the manufacturer’s instructions. PMA (50 ng/ml) and ionomycin (1 μM) were used as positive controls, and complete medium only was used as the negative control. Spots were scanned and quantified by an ImmunoSpot CTL reader.

Steinbuck M.P., Seenappa L.M., Jakubowski A., McNeil L.K., Haqq C.M, & DeMuth P.C. (2021). A lymph node–targeted Amphiphile vaccine induces potent cellular and humoral immunity to SARS-CoV-2. Science Advances, 7(6), eabe5819.

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Multiparameter T Cell Phenotyping

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Spleens and LNs were harvested and passed through a 40μm cell strainer (Corning Inc., USA), followed by lysis of red blood cells via 2-minute incubation with ammonium chloride-potassium (ACK) lysis buffer (Quality Biological). After magnetic enrichment for T cells (see above), approximately 2×10⁷ cells were stained with a fixable live/dead stain (Invitrogen), followed by tetramer staining. Tetramer staining was performed for 35–45 min at room temperature with PE- and APC-conjugated OVA (SIINFEKL):H-2K^b tetramers (NIH Tetramer Core Facility). The cells were then stained for extracellular antibodies for 15–20min at 4^oC. Samples were fixed with the Invitrogen Fix/Perm buffer kit according to the manufacturer’s instruction. Finally, fixed and permeabilized samples were stained for intracellular markers overnight. For phenotypic analysis, samples were acquired via flow cytometry after fixation and intracellular staining. For cell sorting, samples were sorted into RPMI after extracellular staining.

Pollard J., Hynes G., Yin D., Mandal M., Gounari F., Alegre M.L, & Chong A. (2023). Pregnancy programs epigenetic and transcriptional exhaustion in memory CD8+ T cells. Research Square.

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Isolation and Characterization of Tumor-Infiltrating Lymphocytes

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Adhering to a shortened treatment course (GVAX on days 4 and 7; anti-PD-1 and anti-CD137 antibodies and days 4, 6, and 10), tumor-infiltrating lymphocytes (TILs) were recovered from each mouse liver on day 14 following KPC tumor cell inoculation. Each liver with diffused liver metastases throughout the liver was mechanically processed sequentially through 40-μm and 100-μm nylon filters, and the cell suspension was brought to a volume of 25 mL in CTL media. Cell suspension was centrifuged at 500 g for 5 minutes, and cell pellets were suspended in 4 mL of ACK lysis buffer (Quality Biological) for 2 minutes. Lysing was quenched by adding CTL media to a total volume of 50 mL, and cells were centrifuged again at 500 g. Cell pellets were suspended in 6 mL of 80% Percoll (GE Healthcare Life Sciences), overlaid with 6 mL of 40% Percoll, and then centrifuged for 25 minutes at 2400 g with the brake turned off. The resulting lymphocyte layer was removed from the Percoll gradient, suspended in CTL media, and subsequently washed twice with PBS (Gibco). Cells were split into two populations for staining and analysis; one to be stimulated for IFNγ production prior to flow cytometry analysis, and the other left unstimulated for characterization by flow cytometry.

Muth S.T., Saung M.T., Blair A.B., Henderson M.G., Thomas DL I.I, & Zheng L. (2020). CD137 Agonist-based Combination Immunotherapy Enhances Activated, Effector Memory T cells and Prolongs Survival in Pancreatic Adenocarcinoma. Cancer letters, 499, 99-108.

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Adoptive T Cell Transfer in Rag2-/- Mice

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All experiments involving adoptive transfer of lymphoid cells were carried out on a 1:1 donor to recipient basis according to sex. Experiments of adoptive transfer of purified CD4⁺ and CD8⁺ T cells were carried out on a 2:1 donor to recipient basis. Lymph nodes (LNs: cervical, brachial, inguinal and mesenteric) were processed from each mouse in 5 ml DMEM and centrifuged at 400 g (1500 rpm) for 5 min. Red blood cells were removed by using ACK lysis buffer (Quality Biological INC, Gaithersburg, Maryland). The pellet was resuspended with 5 ml PBS and passed through a 40 μm cell strainer to make a single cell suspension. Dead cells were stained with Trypan Blue (Corning Cellgro) and live cells counted using a hemocytometer. For specific CD4⁺ and CD8⁺ reconstitution, T cells were further isolated by negative selection using the EasySep T cell isolation kit specific for CD4⁺ or CD8⁺ T cells (Stem Cell Technologies, Vancouver, BC, Canada) following manufacturer’s guidelines for manual separation. Rag2^−/− mice were reconstituted with 10 million cells in a total volume of 200 μl via tail vein injections and used for flow cytometry analysis at one, two and four weeks after reconstitution and at two and four weeks after for fluorescent microscopy studies.

Song C., Nicholson J.D., Clark S.M., Li X., Keegan A.D, & Tonelli L.H. (2016). Expansion of Brain T cells in Homeostatic Conditions in Lymphopenic Rag2−/− Mice. Brain, behavior, and immunity, 57, 161-172.

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Induction of Murine Arthritis Model

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DMSO (VWR, Radnor, PA, USA), isoflurane (VetOne, Boise, ID, USA), bovine type II collagen in complete Freund’s adjuvant (Hooke Labs, Lawrence, MA, USA), 1X PBS, berberine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), ACK lysis buffer (Quality Biological, Gaithersburg, MD, USA), RPMI 1640 supplemented to 2 mM l-glutamine, 1% v/v penicillin/streptomycin, 1 mM sodium pyruvate, 10 mM HEPES (all from ThermoFisher, Waltham, MA, USA) 0.05 mM β-mercaptoethanol (Bio-Rad, Hercules, CA, USA), and 10% fetal bovine serum (VWR/Seradigm, Radnor, PA, USA).

Vita A.A., Aljobaily H., Lyons D.O, & Pullen N.A. (2021). Berberine Delays Onset of Collagen-Induced Arthritis through T Cell Suppression. International Journal of Molecular Sciences, 22(7), 3522.

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