Ammonium chloride potassium lysis buffer

Manufactured by Lonza

Sourced in Switzerland

Ammonium-chloride-potassium lysis buffer is a reagent used for the lysis of cells, particularly blood cells, in laboratory settings. Its core function is to disrupt cell membranes and facilitate the release of cellular contents for further analysis.

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Lab products found in correlation

Collagenase, by Merck Group (2 mentions) Dispase 2, by Merck Group (2 mentions) M csf, by Thermo Fisher Scientific (2 mentions) Cytoflex, by Beckman Coulter (1 mentions) Gentlemacs tissue dissociator, by Miltenyi Biotec (1 mentions) Wild type c57bl 6j mice, by Jackson ImmunoResearch (1 mentions)

Close spelling variants of this product

Lysis buffer (8 citations) Ammonium-Chloride-Potassium (ACK) lysis buffer (6 citations) Ammonium-chloride-potassium buffer (5 citations) Ammonium chloride buffer (5 citations)

6 protocols using ammonium chloride potassium lysis buffer

Quantifying Macrophage Subsets in Murine Tissues

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Spleen samples from the mice were minced, and single cells were obtained by passing the splenocytes through a 40 μm strainer. Red blood cells were dissolved in an ammonium–chloride–potassium lysis buffer (Lonza, Basel, Switzerland). After washing, single cells were stained with specific antibodies for 30 min on ice to allow for the count of the total number of macrophages (CD11b⁺F4/80⁺), M2a (CD11b⁺F4/80⁺CD163⁻CD206⁺), and M2c (CD11b⁺F4/80⁺CD163⁺CD206⁺) macrophages as well as the evaluation of the M0/M1 (CD11b⁺F4/80⁺CD163⁻CD206⁻) ratio.
Gastrocnemius (GAS) muscle samples were minced and incubated in collagenase and Dispase II (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h. The tissues were dissociated by passing the treated material through a 40 μm strainer. Single cells were stained with specific antibodies for 30 min on ice to permit the total number of macrophages (CD45⁺F4/80⁺) and the number of M1 (CD45⁺F4/80⁺CD86⁺CD206⁻) macrophages to be counted.
A flow cytometry was performed using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed using FlowJo v10 software (Ashland, OR, USA).

Oh H.J., Jin H, & Lee B.Y. (2023). Hesperidin Ameliorates Sarcopenia through the Regulation of Inflammaging and the AKT/mTOR/FoxO3a Signaling Pathway in 22–26-Month-Old Mice. Cells, 12(15), 2015.

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Macrophage Subtype Identification in Tissues

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Spleen samples were minced, and single cells were obtained by passing the products through 40 μm strainers. Red blood cells were lysed in an ammonium–chloride–potassium lysis buffer (Lonza, Basel, Switzerland), and after washing, the cells were incubated for 30 min on ice with specific antibodies to identify M1 (CD45⁺CD206⁻CD86⁺) and M2a/M2c (CD45⁺CD206⁺CD86⁻) macrophages.
Quadriceps and gastrocnemius muscle samples were diced and incubated in collagenase and Dispase II (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C. The tissues were then dissociated by passing them through 40 μm strainers and incubated for 30 min on ice with specific antibodies to identify M1 (CD45⁺CD11c⁺CD206⁻) and M2 (CD45⁺CD11c⁻CD206⁺) macrophages.
Flow cytometry was performed using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were assessed using FlowJo v10 software (Ashland, OR, USA).

Oh H.J., Jin H., Lee J.Y, & Lee B.Y. (2023). Silk Peptide Ameliorates Sarcopenia through the Regulation of Akt/mTOR/FoxO3a Signaling Pathways and the Inhibition of Low-Grade Chronic Inflammation in Aged Mice. Cells, 12(18), 2257.

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Generation of Bone Marrow-Derived Macrophages

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Bone marrow derived macrophages (BMDMs) were generated as previously described (Trouplin et al., 2013 ). In brief, bone marrow was extracted from the tibias and femurs of the hind legs of mice 8–12 weeks of age with a syringe. Afterward, red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Lonza) and the cells were incubated for 4 hours at 37°C with 5% CO2 in a non-tissue culture treated Petri-dish with BMDM media (RPMI supplemented with 10% FBS, 100 U/mL penicillin, 100 μmg/mL streptomycin, 1% sodium pyruvate, 25 mM HEPES buffer, 2mM L-glutamine, and 50 mM 2-mercaptoethanol). After 4 hours, the non-adherent cells were transferred to a new non-tissue culture treated Petri dish and incubated with BMDM media supplemented with 20 ng/mL M-CSF (PeproTech). On day 3 after plating, 10 mL of BMDM media supplemented with 20 ng/mL M-CSF was added to the dish. After 6 days, BMDMs were lifted with PBS+5mM EDTA and gentle scraping. Cell suspensions were seeded onto plates at a density of 250,000 cells/ml for population ELISA experiments or onto the microwell device at a density of 125,000 cells/mL.

Alexander A.F., Kelsey I., Forbes H, & Miller-Jensen K. (2021). Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response. Cell reports, 36(12), 109728.

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Tuberculosis Infection Modeling in Mice

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For Mtb, mice were anesthetized and inoculated intranasally with ∼1,500 CFU of one of the Erdman strains (smyc′::mCherry, hspx′::GFP/smyc′::mCherry, and hsp60′::GFP) resuspended in 30 μl of PBS containing 0.05% Tween 80. Inoculum dosage was confirmed by plating different dilutions of the bacterial stocks in 7H10 agar plates supplemented with OADC Enrichment, glycerol, and hygromycin B. Plates were incubated at 37°C and colonies enumerated 3 wk after. 3 wk post-infection (w.p.i.), mice were sacrificed, and the lungs were aseptically removed and placed in PBS containing 5% FBS. To minimize unwanted changes in the gene expression profile of both host and bacteria, samples were kept on ice and immediately processed using a GentleMACS tissue dissociator (Miltenyi Biotec; Pisu et al., 2020b (link)). The dissociated lung material was then passed through a 70-µM cell strainer, and red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Lonza).
For BCG, mice were intravenously infected with 10⁶ BCG (Pasteur) bacilli. Lung cell suspensions were obtained as detailed above.

Pisu D., Huang L., Narang V., Theriault M., Lê-Bury G., Lee B., Lakudzala A.E., Mzinza D.T., Mhango D.V., Mitini-Nkhoma S.C., Jambo K.C., Singhal A., Mwandumba H.C, & Russell D.G. (2021). Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung. The Journal of Experimental Medicine, 218(9), e20210615.

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Isolation of Bone Marrow Cells

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Fibulas and tibias were removed, and bone marrow cells were isolated using a needle and syringe and PBS. Red blood cells were lysed using ammonium-chloride-potassium lysis buffer (Lonza, Basel, Switzerland), and cells were washed and counted.

Linley H., Ogden A., Jaigirdar S., Buckingham L., Cox J., Priestley M, & Saunders A. (2023). CD200R1 promotes interleukin-17 production by group 3 innate lymphoid cells by enhancing signal transducer and activator of transcription 3 activation. Mucosal Immunology, 16(2), 167-179.

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Isolation of Bone Marrow-Derived Macrophages

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Wild type C57BL/6J mice purchased from Jackson Laboratories were used. Bone marrow-derived macrophages were generated as previously described 43 (link) . Briefly, bone marrow was extracted from the hind legs of the mouse with a syringe. After red blood cell lysis with ammonium-chloridepotassium lysis buffer (Lonza), cells were incubated for 4 hours at 37º C with 5% CO2 in a nontissue culture (TC) treated plastic petri dish with BMDM media (RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1% sodium pyruvate, 25 mM HEPES buffer, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol). After 4 hours, the non-adherent cells were transferred to a new petri dish and incubated with BMDM media + 20 ng/ml macrophage-colony stimulating factor (M-CSF; Peprotech). After 3 days, an additional 10 ml of BMDM media + 20 ng/ml M-CSF was added to the plate. 6 days after plating, cells were harvested in PBS + 5 mM EDTA with gentle scraping, and the cell suspension was used to seed new non-TC treated dishes or microwell devices. All mice were housed in the Yale Animal Resources Center in specific pathogen-free conditions. All animal experiments were performed according to the approved protocols of the Yale University Institutional Animal Care and Use Committee.

Muñoz-Rojas A.R., Kelsey I., Pappalardo J.L., & Miller‐Jensen K. (2020). Co-stimulation with opposing macrophage polarization cues leads to orthogonal secretion programs in individual cells.

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