Harvested lung lobes from infected and uninfected mice were washed with sterile 1× DPBS following organ dissection and then digested with collagenase IV (100 U/ml in RPMI; Sigma-Aldrich) at 37°C for 45 min under agitation (200 rpm). The enzymatic reaction was stopped by adding FBS. The pulmonary cells were dispersed by passage through a 100-µm pore-size cell strainer and red blood cells depleted by the addition of ammonium-chloride-potassium lysis buffer (Gibco) at room temperature for 2 min. Cells were next washed by centrifugation (1,200 rpm for 5 min) and cell pellets resuspended in RPMI supplemented with 10% FBS and 25 µg/ml gentamicin. The resulting cell preparations were counted and then seeded (8 × 104 cells/well) in round-bottom 96-well plates for LAA, as well as cell surface and Live/Dead staining.
Ammonium chloride potassium lysis buffer
Manufactured by Thermo Fisher Scientific
Ammonium-chloride-potassium lysis buffer is a solution used to lyse red blood cells during the isolation of white blood cells or other cells from whole blood samples. It is a hypotonic solution that causes the selective lysis of red blood cells, while leaving the white blood cells intact for further analysis or processing.
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Lab products found in correlation
Dnase 1, by Merck Group (3 mentions)
Collagenase 4, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cytation 5 reader, by Agilent Technologies (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
40 μm nylon cell strainer, by BD (1 mentions)
70 μm nylon cell strainer, by BD (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Cell strainer, by Greiner (1 mentions)
Dnase 1, by Roche (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
C tube, by Miltenyi Biotec (1 mentions)
Collagenase d, by Roche (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
70 m cell strainer, by Greiner (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
21 protocols using ammonium chloride potassium lysis buffer
1
Lung Cell Isolation and Characterization
2
Isolation and Analysis of Human Leukocytes and Neutrophils
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Human whole leukocytes were isolated by lysing erythrocytes with ammonium-chloride-potassium lysis buffer (Gibco). Human neutrophils were isolated by density gradient centrifugation using the Polymorphprep kit according to the manufacturer’s instructions (Axis-Shied). Cells were subsequently washed with phosphate-buffered saline (PBS) and resuspended in phenol red-free RPMI 1640 medium supplemented with 2% fetal bovine serum (Gibco). For cytotoxicity assays, whole leukocytes were dispensed at 2 × 105 cells/well in a final volume of 100 μl/well in a 96-well plate. Exoprotein samples described above were added to the cells and incubated for up to 1 h at 37°C. PI (10 μg/ml) was added, and the cells were further incubated for 15 min. The cytotoxicity was measured by the fluorescence signal at 590 nm using a NovoCyte flow cytometer (Agilent Technologies) and a Cytation 5 reader (BioTek). For Transwell experiments, human HEp2 cells were cultured in 24-well plates to 80% confluence. A 0.45-μm Transwell insert (Corning) containing human neutrophils at 1 × 105 cells/well was placed in the 24-well plate. In some experiments, LukF and LukS (1 μg of each; IBT Bioservices) were added to the Transwells and incubated for up to 12 h. The cytotoxicity was monitored using a Cytation 5 reader (BioTek).
3
Tissue Disruption and Cell Isolation
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Spleens and lymph nodes were disrupted by mashing through a 40-μm nylon cell strainer (BD Biosciences, San Jose, CA) using a sterile 2-ml syringe plunger. Tumours were excised and chopped into pieces using scalpel blades. The pieces were then mashed with a syringe plunger and the resulting cell suspension was filtered through 70-μm nylon cell strainers (BD Biosciences). The cell suspensions were then centrifuged before red blood cells lysis using ammonium–chloride–potassium lysis buffer (Gibco, Grand Island, NY).
4
Extraction of Tumor-Infiltrating Lymphocytes from Melanoma
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To obtain tumor-infiltrating lymphocytes (TILs) from C57BL/6J mice bearing established subcutaneous B16 melanoma, solid tumors were excised, chopped finely using tweezers and scissors, and digested with 1 mg/ml collagenase D (Sigma; #11088866001) and 1000 IU/ml DNAse I (Sigma; #4716728001) for 60 min at 37 °C. Following digestion, the fragments were filtered through 40 μm cell strainers (Greiner Bio-One). After removal of red blood cells with ammonium-chloride-potassium lysis buffer (Gibco; A10492), the cells were resuspended in 4 ml RPMI medium and layered carefully over 4 ml Ficoll-paque (GE Healthcare; #17-1440-02), followed by centrifugation at 1025g for 20 min at room temperature. The enriched TILs, obtained at the interface as a thin buffy layer were washed with PBS and finally resuspended in FACS staining buffer for further staining procedures. For each mouse, lymphocytes from spleen were also harvested.
5
Isolation and Processing of Murine Immune Cells
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Spleens were aseptically removed and made into single cell suspensions. Lungs were perfused with PBS, aseptically removed, and digested into a single cell suspension as previously described 33 (link). Red blood cells were lysed using ammonium chloride potassium lysis buffer (Gibco) and washed with RPMI 1640 supplemented with 10% fetal calf serum (Atlas), L-glutamine, sodium pyruvate, and β-mercaptoethanol. The total number of viable cells was determined using a hemocytometer by trypan blue exclusion.
6
Bone Marrow Extraction for Trauma Research
7
Isolation and Analysis of Mouse Lung Cells
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hUC-MSCs were suspended in ice–cold phosphate-buffered saline (PBS) and were kept on ice until administration. Mice received an IV injection of 2.5 × 105 untransduced hUC-MSCs or PBS (100 μl) under anesthesia with isoflurane. Two or twenty-four hours postinjection, the animals were culled by cervical dislocation. The lungs were removed en bloc. The large airways were dissected from the peripheral lung tissue and each lung lobe was separated. The lung lobes were cut into small pieces with scissors, transferred into C-tubes (Miltenyi Biotec), and processed in digestion buffer (1 mg/ml of Collagenase D and 80 U/ml DNase I, both from Roche, in DMEM) and a GentleMACS dissociator (Miltenyi Biotec), according to the manufacturer's instructions. The lung homogenates were strained through a 70 mm nylon mesh to obtain single-cell suspensions. Red blood cells were lysed using ammonium–chloride–potassium lysis buffer (Gibco, A1049201). The resultant cells were counted using an automated cell counter (TC10, BioRad).
8
Isolation and Characterization of Adipocytes and Stromal Vascular Cells
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The collected visceral fat pads were pooled before further experiments (CON: 3, OB: 2, OBA: 2 pooled). The fat tissues were cut into small pieces with scissors and incubated in a shaking water bath for 40 min (37°C, 170 cycle/min) with Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 0.1% collagenase type 2 (Sigma-Aldrich) and 2% BSA. The digested suspension was passed through a 200 μm pore nylon mesh to remove the tissue debris. The filtrate was centrifuged at 500 × g for 5 min at RT, and the top layer was transferred to a new tube as adipocytes. The cell pellet (SVCs) at the bottom was incubated with 3 ml of ammonium-chloride-potassium lysis buffer (Gibco) for 3 min at RT to remove the red blood cells. The isolated adipocytes and SVCs were washed twice with DMEM/10% fetal bovine serum (FBS) and used for flow cytometry or cell culture.
9
Spleen Cell Isolation and Lysis
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Spleens were mechanically homogenized on a 70-µm cell strainer (Greiner Bio-One) with the plunger of a 2-mL syringe. Cell pellets of spleens were resuspended in 2 mL of HBSS (w/o Ca/Mg) and incubated with 5 mL of ammonium-chloridepotassium lysis buffer (Gibco) for 10 minutes at 4 °C for erythrocyte lysis. Cells were washed with HBSS and resuspended in FACS buffer for FACS staining.
10
Peritoneal and Bone Marrow-Derived Macrophage Isolation
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As previously described, murine peritoneal and BMDMs were prepared for in vitro assays52 (link). Peritoneal macrophages were collected as described below. Brewer’s thioglycolate medium (BD Diagnostic Systems, Sparks, MD) was injected into the murine peritoneal cavity 3 days before macrophage collection. Ice-cold phosphate-buffered saline (PBS) was injected into the peritoneal cavity, and cells were harvested. Red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Thermo Fisher Scientific Inc., Cleveland, OH). Then, the cells were washed with PBS, resuspended in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma–Aldrich) and 1% penicillin/streptomycin and plated in culture plates. One hour later, the nonadherent cells were removed, and the remaining adherent cells were cultured further in fresh medium. Bone marrow-derived macrophages were harvested from the tibiae and femurs of donor mice. After red blood cell lysis and washing, the cells were resuspended and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (R&D Systems, Minneapolis, MN).
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