Each cell line (KP1-KP3), was derived from a different primary sarcoma generated from individual KP mice. Sarcomas between 200 and 300 cm3 were harvested in cell culture hoods and cut into very small (approximately 0.25×0.25×0.25 cm) pieces. Then, they were digested using a solution containing 5 mg/ml collagenase, 2.4 U/ml dispase and 0.05% trypsin in phosphate buffer saline (PBS) for a minimum of 1 hour. The digested mixture/slurry was then filtered using a 70 μm filter followed by lysis of red blood cells using ammonium chloride/potassium buffer according to the manufacturer's protocol (Lonza). Lastly, cells were filtered using a 40 μm sieve and cultured for 5-8 passages to deplete stromal cells before they were used for the experiments. Cells were cultured in DMEM supplemented with 10% FBS and incubated at 37°C with 5% CO2 in a humidified cell culture incubator.
Ammonium chloride potassium buffer
Manufactured by Lonza
Sourced in United States
Ammonium-chloride-potassium buffer is a laboratory buffer solution used to maintain a specific pH level in chemical and biological experiments. It is a mixture of ammonium chloride, potassium chloride, and other components that work together to stabilize the pH of the solution.
Automatically generated - may contain errors
Lab products found in correlation
Wallac liquid scintillation counter, by PerkinElmer (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Mouse neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Perm wash buffer, by BD (1 mentions)
Anti cd3ε clone 145 2c11, by BD (1 mentions)
Rat anti mouse cd16 cd32 monoclonal antibody, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Collagenase 2 and 3, by Worthington (1 mentions)
Dnase 1, by Roche (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Lonza (1 mentions)
5 protocols using ammonium chloride potassium buffer
1
Primary Sarcoma Cell Line Derivation
2
Lymphocyte Proliferation Assay in Mice
3
Isolation of Neutrophils from Mouse Bone Marrow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57/B6 wild-type and Fpr1/2 double mutant (Fpr1–/– and Fpr2–/–) mice were from Ji Ming Wang’s lab, National Cancer Institute, National Institutes of Health (NIH). Bone marrow was collected from the hind legs. Briefly, one end of the bone was cut open, and bone marrow was collected by centrifugation at 8600 × g for 10 s. Red blood cells were removed with ammonium-chloride-potassium buffer (Lonza) at room temperature. After the cells were counted, cells were subject to neutrophil isolation using a mouse neutrophil isolation kit (Miltenyi Biotec; order no. 130097658). Collected neutrophils were stored in PBS supplemented with 2% FBS and 2 mM EDTA at 4°C. The purity of neutrophils was determined by staining the cells with mouse Gr-1/Ly-6G PE-conjugated antibody and CD11b FITC-conjugated antibody (R&D Systems). The purity was above 95% (unpublished data).
4
Characterizing Immune Cell Populations in Murine Tumor Models
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens and draining lymph nodes (DLNs) from 4T1 and 4T1.GCSF− tumor-bearing mice were isolated when tumors reached 500–700 mm3. Single cell suspensions were prepared by mechanically dissociating spleen and DLNs with a syringe plunger and passing samples through a 40-μm nylon mesh cell strainer. Splenocytes were additionally treated with ammonium-chloride-potassium buffer (Lonza, Allendale, NJ, USA) for 10 min to lyse red blood cells. Single cell suspensions were then blocked with purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Biosciences) and stained with fluorochrome-conjugated anti-CD11b (clone M1/70), anti-CD19 (clone 1D3), anti-Ly6G and Ly6C (clone RB6-8C5), anti-CD25 (clone PC61), anti-CD4 (clone GK1.5), and anti-CD3ε (clone 145-2C11) (BD Biosciences).
Cells were then rinsed, fixed and permeabilized with 1× Perm/Wash buffer from BD Biosciences. The permeabilized cells were further stained with fluorochrome-conjugated anti-FoxP3 and read on a BD FACSCanto II flow cytometer. Frequencies of MDSCs, T cells, B cells, and regulatory T cells (Tregs) in the single cell suspensions were determined using FlowJo software (Tree Star, San Carlos, CA, USA). For mice bearing 4T07, 67NR, 66Cl4 and 168FARN tumors, only the spleens were isolated and stained for MDSCs.
Cells were then rinsed, fixed and permeabilized with 1× Perm/Wash buffer from BD Biosciences. The permeabilized cells were further stained with fluorochrome-conjugated anti-FoxP3 and read on a BD FACSCanto II flow cytometer. Frequencies of MDSCs, T cells, B cells, and regulatory T cells (Tregs) in the single cell suspensions were determined using FlowJo software (Tree Star, San Carlos, CA, USA). For mice bearing 4T07, 67NR, 66Cl4 and 168FARN tumors, only the spleens were isolated and stained for MDSCs.
5
Isolation of Myeloid and Lymphoid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Myeloid cells and lymphocytes (include T cells and innate lymphoid cells) were isolated from tissues including mLN, SI, and LI. For the mLN, the tissues were grounded and homogenized in a 70-μm cell strainer. After spinning down, red blood cells were removed by the addition of ammonium-chloride-potassium buffer (Lonza) for 10 min on ice. Subsequently, the single-cell suspension was washed with Dulbecco's PBS containing 0.5% bovine serum albumin (Thermo Fisher Scientific) and 2 mM EDTA (Lonza). For the gut, intestines were cut open longitudinally, the tissues were fit, and Peyer’s patches were removed. Intestines were then washed with cold PBS and cut into pieces. Epithelial layers were removed by shaking incubation in 5 mM EDTA (Lonza) Ca2+ and Mg2+ free 1640 medium (Life Technologies) for 30 min each at 37°C. Then, the intestines were cut into fine pieces and digested twice for 40 min each at 37°C with Collagenase II and III (1 mg/ml; Worthington), and DNase I (200 μg/ml; Roche). Cells were isolated with 30–60% Percoll gradient and washed twice with cold Dulbecco's PBS.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!