Tumors from control, Abseff and Absineff group were selected for tumor extraction. The single-cell suspension of mouse splenocytes was prepared by filtering through a 100-mm and 40-mm cell strainer (SPL Life Sciences, Pocheon , Korea) while red blood cells (RBCs) were removed with ammonium-chloride-potassium lysing buffer (Lonza, Basel, Switzerland). Further, tumors were digested with collagenase IV (0.2 mg/mL, Sigma-Aldrich) and DNase I (0.02 mg/mL, Sigma-Aldrich) for 30 min. Tumor tissues were then squashed through 70 m cell strainers with a syringe. TILs were extracted from the central layer of a 40–80% Percoll (Sigma-Aldrich) gradient centrifugation (run at 800 g for 20 min at room temperature without deceleration). CytoFLEX flow cell counter (Beckman) was used to examine TILs such as CD4+/CD5+, CD3+, CD8+/CD3+, CD25+/CD4+, CD45+, CD103+/CD8+, CD39+/CD8+, CD11c+, F4/80+, NK1.1+, CD86+, CD68+, and CD206+.
Ammonium chloride potassium lysing buffer
Manufactured by Lonza
Sourced in United States, Switzerland
Ammonium-chloride-potassium lysing buffer is a solution designed for the lysis of red blood cells. It functions by causing the osmotic lysis of red blood cells, allowing for the isolation and analysis of other cell types, such as white blood cells, from a sample.
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Lab products found in correlation
Histopaque, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Lonza (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Cytoflex flow cell counter, by Beckman Coulter (1 mentions)
Percoll, by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase type ia solution, by Merck Group (1 mentions)
Macs buffer, by Miltenyi Biotec (1 mentions)
70 m nylon cell strainer, by BD (1 mentions)
Cd3 145 2c11, by BD (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd19 1d3, by BD (1 mentions)
Cd21 7g6, by BD (1 mentions)
Igm 2 41, by BD (1 mentions)
Cd23 b3b4, by BioLegend (1 mentions)
Cd93 aa4 1, by BD (1 mentions)
Dnase, by Roche (1 mentions)
70 μm nylon cell strainer, by BD (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
19 protocols using ammonium chloride potassium lysing buffer
1
Tumor Infiltrating Lymphocytes Isolation
2
Isolation of Adipose-Derived Stromal Cells
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All protocols were reviewed and approved by the Seoul National University Hospital Institutional Review Board. Human subcutaneous and visceral adipose tissues were surgically obtained from four patients with benign urologic or gynecologic diseases. The adipose tissues were dissociated (37°C, 1 hour) with collagenase type IA solution (Sigma-Aldrich, St. Louis, MO). The cells were centrifuged (500 ×g, 4 minutes) and the stromal vascular fraction (SVF) was collected as a pellet. The SVF was treated with ammonium-chloride-potassium lysing buffer (Lonza, Muenchsteiner-strasse, Switzerland) at room temperature for 2 minutes, and then centrifuged. The resulting cell pellet was diluted with a magnetic-activated cell sorting (MACS) buffer (MiltenyiBiotec, Teterow, Germany). To avoid contamination with hematopoietic stem cells and endothelial cells, the cells were incubated (room temperature, 15 minutes) with anti-CD45 and anti-CD31 antibodies attached to the microbeads (BD Biosciences, San Jose, CA). The CD45- and CD31-negative cells were collected from the SVF using MACS (Fig. 1A ).
3
Isolation of Murine Lung Tumor Cells
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Primary lung tumor cell cultures were derived from the lungs of one control (LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/−;TetO-Cre+/−) and one Nanos3 (Nanos3LSL/−;LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/−;TetO-Cre+/−) NSCLC mouse. After dissection, the lungs were incubated in PBS with geneticin (250 μg/ml) for 1 h at room temperature. The lungs were fragmented and dissociated by sterile methods. Dissociation was at 37 °C for 2 to 3 h in DMEM containing 250 μg/ml gentamycin, 0.5% glucose, 0.125 units/ml dispase II, 0.2% collagenase, and 10% FCS. The resulting cell suspension was consecutively run through 70-μm and 40-μm cell strainers, and then centrifuged at 400 x g for 7 min at 4 °C. Cell pellets were suspended in 1 ml of Ammonium-Chloride-Potassium lysing buffer (Lonza) for 5 min. Cells were washed with PBS and seeded in RPMI medium supplemented with 10% FCS and non-essential amino acids.
4
PBMC Isolation from Whole Blood
5
Isolation and Enrichment of Thymocytes and Splenocytes
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TECs were isolated and enriched as previously described (20 (link)). For thymocyte isolation, thymi were harvested and disrupted with a 1-ml syringe plunger through a 70-µm nylon cell strainer (BD Biosciences) and filtered to obtain a single-cell suspension using PBS containing 2% FBS and 0.01% sodium azide. Spleens were harvested and processed to obtain single-cell suspensions using the frosted ends of microscope slides in PBS containing 2% FBS and 0.01% sodium azide. Spleens were incubated with ammonium chloride potassium lysing buffer (Lonza) for 3 min at room temperature to lyse RBCs. Cells were rinsed with PBS containing 2% FBS and 0.01% sodium azide.
6
Mouse Spleen Cell Isolation Protocol
7
Isolation of Tumor-Infiltrating Lymphocytes
8
Opsonization and Phagocytosis Assay with SRBCs
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Sheep red blood cells (SRBCs) (Lampire Biologicals, Pipersville, PA, USA) were washed several times with saline and opsonized by incubation with rabbit anti-SRBC IgG (2 μL per 1 × 109 cells in 1 mL) (MP Biomedicals, Santa Ana, CA, USA) at 37 °C for 1 h with gentle agitation. RBCs were washed with saline to remove the unbound antibody. Macrophages cultured in 24-well plates at 1 × 105 cells per well for 5 d were incubated with opsonized RBC at a ratio of 25:1 at 37 °C for 1.5 h. After incubation, the cultures were washed to remove excess RBC and ammonium-chloride-potassium lysing buffer (Lonza, Walkersville, MD, USA) was added to remove RBC attached to the surface of the macrophages. A minimum of 300 macrophages per field was scored for the presence of ingested RBC. Non-opsonized RBCs were used as a negative control.
9
S. aureus Infection Model in Mice
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Pathogen-free 10-week-old C57BL/6 female mice were purchased from Charles River (Germany) and maintained according to institutional guidelines in individually ventilated cages with food and water provided ad libitum. Mice were intravenously inoculated with 106 CFU of S. aureus in 100 μl of PBS via a lateral tail vein, and sacrificed by CO2 asphyxiation at day 21 after bacterial inoculation. The spleen was removed from infected mice and single-cell suspensions were prepared by gently teasing the spleen tissue through a 100 µm pore size nylon cell strainer and the bone marrow was flushed out of both tibia and femur. Spleen and bone marrow cells were spun down, erythrocytes were lysed after incubation for 5 min at RT in ammonium-chloride-potassium lysing buffer (Lonza), washed with PBS + 10% FCS and resuspended in RPMI-1640 medium (Gibco) supplemented with 10% FCS and antibiotic–antimycotic (VWR International).
10
Isolation of Mouse Blood Cells
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Blood was collected into EDTA-coated syringes by cardiac puncture from sacrificed mice. Suspensions were washed and resuspended in ammonium-chloride-potassium lysing buffer (Lonza) for 3 min on ice twice. Suspensions were then washed and resuspended in media containing 10% FCS until staining.
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