Female and/or male mice at 8–12 weeks of age were used. NOD scid gamma (NSG) mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were purchased from the Jackson Laboratory and bred in our facility. Mice were intravenously injected with 5×105 Raji-GFP/FFluc or Nalm6-GFP/FFluc cells at week −1 and 2×106 CAR T cells were intravenously injected at week 0. For bi-specific CAR T cells mice received 1×106 CAR T cells. Tumors were measured using an IVIS Lumina III In Vivo Imaging System (PerkinElmer) for bioluminescence imaging (BLI) weekly. Mice were monitored for illness daily and sacrificed when there was evidence of leukemia progression, such as decreased activity, hunched posture, or ruffled coat. At certain time points, blood was collected from the submandibular vein into tubes containing K3 EDTA (Sarstedt). At mouse sacrifice blood, spleen, and/or femurs were collected. For blood samples red blood cells were lysed using ammonium-chloride-potassium buffer (ThermoFisher) and stained for flow cytometry as described below. Bone marrow was isolated from femurs by cutting both ends of the femur and flushing it with a syringe containing phosphate buffered saline (PBS) (Gibco). These cells were passed through a 70 µm cell strainer. Red blood cells were lysed as described above and stained for flow cytometry (as described below).
Ammonium chloride potassium buffer
Manufactured by Thermo Fisher Scientific
Ammonium-chloride-potassium buffer is a laboratory solution used to maintain a specific pH range in various applications. It is a mixture of ammonium chloride, potassium chloride, and other compounds. This buffer solution is commonly used in biological and biochemical experiments to control and stabilize the pH of the system under investigation.
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Lab products found in correlation
Liberase tl, by Merck Group (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Cellometer auto 2000 cell counter, by Revvity (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Roche (2 mentions)
100 μm filters, by Thermo Fisher Scientific (2 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nod cg prkdcscid il2rgtm1wjl szj, by Jackson ImmunoResearch (1 mentions)
Ivis lumina 3 in vivo imaging system, by PerkinElmer (1 mentions)
K3 edta, by Sarstedt (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Roche (1 mentions)
Collagenase a, by Roche (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
10 protocols using ammonium chloride potassium buffer
1
Evaluating CAR T Cell Efficacy
2
Culturing Diverse Cancer Cell Lines
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Human colorectal carcinoma HCT116 cell lines with or without wild-type p53 expression (p53+/+ and p53−/−), human invasive breast cancer MDA-MB-231 cells, human THP-1 monocytes and murine macrophage-like RAW264.7 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), L-glutamine (2 mM) (Thermo Fisher Scientific), penicillin (100 units/mL) and streptomycin (100 μg/mL) (Thermo Fisher Scientific) in a 37 °C incubator with 5% CO2 and 95% humidity.
Colorectal cancer from the patient was minced and dissociated with 200 U/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and passed through a Falcon 40 M cell strainer (Corning, Corning, NY, USA). Red blood cells were lysed with ammonium–chloride-potassium buffer (Thermo Fisher Scientific). The isolated primary cancer cell line (C6P2-L1) was first grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% FBS, L-glutamine (2 mM), penicillin (100 units/mL) and streptomycin (100 μg/mL) and then subcultured in DMEM supplemented with 10% FBS in a 37 ℃ incubator with 5% CO2.
Colorectal cancer from the patient was minced and dissociated with 200 U/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and passed through a Falcon 40 M cell strainer (Corning, Corning, NY, USA). Red blood cells were lysed with ammonium–chloride-potassium buffer (Thermo Fisher Scientific). The isolated primary cancer cell line (C6P2-L1) was first grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% FBS, L-glutamine (2 mM), penicillin (100 units/mL) and streptomycin (100 μg/mL) and then subcultured in DMEM supplemented with 10% FBS in a 37 ℃ incubator with 5% CO2.
3
Isolation of Immune Cells from Tissues
4
Tumor Immune Cell Isolation and Analysis
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Tumors, lymph nodes, and spleens were finely minced and incubated at 37 °C at 250 rpm in RPMI supplemented with 2% FBS, 0.5 mg/mL Collagenase A (Roche), and 0.1 mg/mL DNase1 (Roche) for 30 min (tumor) or 15 min (lymph nodes and spleen). After incubation, single suspensions were filtered through a 70-μm mesh. Remaining erythrocytes were lysed in ammonium–chloride–potassium buffer (Life Technologies). Circulating leukocytes were obtained from ammonium–chloride–potassium–treated whole-blood samples. Single-cell suspensions were stained with fluorochrome-conjugated antibodies and MHC tetramers for flow cytometry analysis of T cell responses and for the characterization of the tumor leukocyte infiltrate (SI Appendix, Materials and Methods ). Single-cell suspensions were incubated with the indicated peptide epitopes (SI Appendix, Table S1 ) for in vitro, antigen-specific T cell stimulation (SI Appendix, Materials and Methods ) for intracellular cytokine staining or ELISPOT. For details, see SI Appendix, Materials and Methods .
5
Metabolite Extraction from Immune Cells
6
Perfusion and Tissue Sampling Protocol
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Before initiation of perfusion, perfusate samples from retrograde flush with cold phosphate-buffered saline (about 300 mL) and tissue samples from wedge biopsies (about 3 g) were obtained. The samples collected prior to initiation of perfusion were labeled as 0th hour. During perfusion, perfusate samples (about 300 mL) and tissue samples (about 3 g) were obtained at each time point (1st hour, 3rd hour, and 6th hour of perfusion). Perfusate samples were spun down at 400G and treated with ammonium-chloride-potassium buffer (Gibco|Thermo Fisher Scientific, Waltham, MA) to lyse remaining red blood cells. Liver tissue was mechanically disrupted and enzymatically digested at 37°C for 90 minutes using 100U/mL of Type II collagenase (Gibco|Thermo Fisher Scientific, Waltham MA) in Hank’s Balanced Salt Solution (Gibco|Thermo Fisher Scientific, Waltham MA) prior to filtration through 100-micron filters (Fisher Scientific, Waltham, MA). Samples were then washed and resuspended in phosphate-buffered saline.
7
Isolation of Murine Lung Immune Cells
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33 mg/ml Liberase TL and 0.1 mg/ml DNase I (both from Sigma-Aldrich) at 37°C for 45 min under agitation (200 rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70-μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 5 min at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using ViaStain acridine orange propidium iodide staining on a Cellometer Auto 2000 Cell Counter (Nexcelom). For assessment of cytokine production, single-cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1× Protein Transport Inhibitor Cocktail (eBioscience) for 5 h.
8
Lung Dissociation for Single-Cell Analysis
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33mg/mL Liberase TL and 0.1mg/mL DNase I (both from Sigma Aldrich) at 37°C for 45 minutes under agitation (150rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 3 minutes at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using AOPI staining on a Cellometer Auto 2000 Cell Counter (Nexcelom).
9
Lung Tissue Dissociation and Cytokine Analysis
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Lung lobes were diced into small pieces and incubated in RPMI containing 0.33mg/mL Liberase TL and 0.1mg/mL DNase I (both from Sigma Aldrich) at 37°C for 45 minutes under agitation (200rpm). Enzymatic activity was stopped by adding FCS. Digested lung was filtered through a 70μm cell strainer and washed with RPMI. Red blood cells were lysed with the addition of ammonium-chloride-potassium buffer (Gibco) for 5 minutes at room temperature. Cells were then washed with RPMI supplemented with 10% FCS. Live cell numbers were enumerated using AOPI staining on a Cellometer Auto 2000 Cell Counter (Nexcelom).
For assessment of cytokine production, single cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1X Protein Transport Inhibitor Cocktail (eBioscience) for 5 hours.
For assessment of cytokine production, single cell suspensions were incubated in FCS-supplemented RPMI in the presence of 1X Protein Transport Inhibitor Cocktail (eBioscience) for 5 hours.
10
Lung Tissue Sampling during Ex Vivo Perfusion
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Before initiation of perfusion (0th hour), perfusate samples from retrograde flush with cold phosphate‐buffered saline and tissue samples via wedge biopsies were obtained. During perfusion, perfusate samples (about 300 ml) and tissue samples from periphery of lobes (about 500 mg–1500 mg) were obtained at the 1st, 3rd, and 6th hour of perfusion. Perfusate samples were spun down at 400 G and treated with ammonium‐chloride‐potassium buffer (Gibco|Thermo Fisher Scientific, Waltham, MA) to lyse remaining red blood cells. Lung tissue was mechanically disrupted and enzymatically digested at 37°C for 90 min using 100 U/ml Type I collagenase (Gibco|Thermo Fisher Scientific, Waltham MA) in Hank's Balanced Salt Solution (HBSS) prior to filtration through 100‐micron filters (Fisher Scientific, Waltham, MA). Samples were then washed and resuspended in phosphate‐buffered saline.
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