Lung tissues were excised completely and minced in serum-free RPMI 1640 medium under aseptic conditions and incubated with 0.4 mg/ml collagenase IV (LS004186, Worthington) and 1U/ml DNase I (Roche) for 3 h at 37 °C. Cell suspensions were filtered through a series of nylon meshes ranging from 70-mm to 40-mm and washed with PBS. Lymphocyte-enriched cell suspension was acquired by percoll density gradient (70% and 30%, GE Healthcare) centrifugation. Cells were stained for flow cytometric analyses.
Ls004186
Manufactured by Worthington
Sourced in United States
LS004186 is a lab equipment product. It is a precision instrument designed for use in scientific research and laboratory settings. The core function of this product is to perform specific tasks required for laboratory experiments and analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Percoll density gradient, by GE Healthcare (2 mentions)
Dnase 1, by Solarbio (2 mentions)
Facsaria 2 cell sorter, by BD (2 mentions)
Dnase 1, by Roche (1 mentions)
Ls002594, by Worthington (1 mentions)
7 aad, by BioLegend (1 mentions)
Cd45.2 apc clone 104, by BioLegend (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd45.2 apc, by BioLegend (1 mentions)
7 protocols using ls004186
1
Isolation of Lung Lymphocytes
2
Primary Murine LADC Cell Isolation
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Primary murine LADC cells were obtained as previously described (41 (link)). Lung lobes were dissected out and immediately placed in 4 mL of medium A (AdMEM/F12, B27, N2, 2% FCS, 1% penicillin/streptomycin, 10 μg/mL insulin, 20 ng/mL EGF, 20 ng/mL FGF, 100 μg/mL Primocin) into GentleMACS C tubes and subjected to automated homogenization using GentleMACS dissociators at Tumor 2 mode, followed by addition of 300 U/mL collagenase IV (Worthington, LS004186) and hyaluronidase 300 U/mL (Worthington, LS002594), as well as incubation for 20 minutes at 37°C with constant shaking. Homogenized lungs were subjected to further dissociation at Tumor 3 mode, followed by centrifugation at 150g for 5 minutes at 4°C. The pellet was resuspended in medium A, and cells were plated onto Ultra-low attachment plates and placed in a humidified incubator at 37°C, 5% CO2. Ninety-six hours later, the medium was replaced with DMEM (10% FCS, 1% penicillin/streptomycin), and cells were replated on standard tissue culture plates. All cell lines were tested as mycoplasma negative by PCR assay.
3
CXCR4 Antagonism Assay Using U2OS-EGFP Cells
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The CXCR4 antagonism was determined by CXCR4 redistribution assay as described previously25 (link). In brief, 5 × 104 U2OS-EGFP cells were seeded in 96-well black plates and cultured to the density of cells above 60%. After washing twice with assay buffer (DMEM medium with 1% FBS and 10 mmol/L HEPES), IV collagenase (0.25 units/mL, LS004186, Worthington, US), HPD@DOX/siPAI-1, HPA/siPAI-1 and HPA@DOX/siPAI-1 (100 nmol/L siPAI-1) were added to the well (30 min). Then, a ten-fold dosage of SDF-1 was added to each well for 1 h. The U2OS-EGFP cells were washed for 4 times with assay buffer and then fixed with 4% paraformaldehyde. Then, the nuclei of cells were stained with DAPI (blue) for 15 min and imaged them using EVOS fluorescence microscopy (EVSO FL, Thermo Fisher, US).
4
Comprehensive Tumor Immune Profiling by scRNA-seq
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Young and old mice were injected subcutaneously with 2×105 B16F10 cells per mouse. The tumors were visible 5 days after engraftment and then the volumes were measured every 2 days. Tumor volumes were determined by caliper measurement using the formula V = length×width2/2. Finally, tumor tissues were excised and weighed at indicated time point (17 days after engraftment). Next, freshly isolated tumor tissues were minced into approximately 1 mm3 cubic pieces and digested using 0.1% collagenase IV (LS004186, Worthington), 0.002% DNAse I (D8071, Solarbio), and 0.01% hyaluronidase (H3506-1G, SIGMA), then incubated on a rocker at 37°C for 40–50 min (according to tumor size). The digested cells were filtered through a 70 µm cell strainer and washed twice with cold PBS (phosphate-buffered saline) added 2% FBS (Fetal bovine serum) buffer. The remaining cells were stained with APC-CD45.2 (Clone 104; BioLegend) and 7AAD (Part 76332; Lot B226294 Biolegend) for 30 min at 4°C, then washed and suspended in PBS (2% FBS) buffer for flow cytometric sorting using FACS Aria II Cell Sorter (BD Biosciences). Sorted CD45.2+7AAD- cells with a purity greater than 95% and viability higher than 90% were used for 10X genomics scRNA-seq.
5
Isolation of Kidney Lymphocytes
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Blood samples of different mice (100 μl) were collected before sample harvest, and then red blood cell lysis buffer (C3702, Beyotime Biotochnology) was used to remove red cells. The washed cells were used for flow analysis.
Kidney tissues were excised completely, minced in serum-free RPMI 1640 medium under aseptic conditions and then incubated with 0.4 mg/ml collagenase IV (LS004186, Worthington) for 1 h at 37°C. Cell suspensions were filtered through a series of nylon meshes and washed with PBS. Lymphocyte-enriched cell suspensions were acquired by Percoll density gradient (70 and 30%, GE Healthcare) centrifugation. Cells were stained for flow cytometric analyses.
Kidney tissues were excised completely, minced in serum-free RPMI 1640 medium under aseptic conditions and then incubated with 0.4 mg/ml collagenase IV (LS004186, Worthington) for 1 h at 37°C. Cell suspensions were filtered through a series of nylon meshes and washed with PBS. Lymphocyte-enriched cell suspensions were acquired by Percoll density gradient (70 and 30%, GE Healthcare) centrifugation. Cells were stained for flow cytometric analyses.
6
Flow Cytometry Analysis of Lung Tumors
7
Isolation and Profiling of Tumor-Infiltrating Immune Cells
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Two groups of mice were injected subcutaneously with 2x10 5 B16F10 cells per mouse. After 5 days, tumor tissues were excised and weighed at indicated time points (every two days).
Tumor volumes were determined by caliper measurement using the formula V = (length × width 2 ) / 2. Freshly isolated tumor tissues were minced into approximately 1 mm 3 cubic pieces and digested using 0.1% collagenase IV (LS004186, Worthington), 0.002% DNAse I (D8071, Solarbio), and 0.01% hyaluronidase (H3506-1G, SIGMA), then incubated on a rocker at 37°C for 40-50 min. The digested cells were filtered through a 70 μm cell strainer and washed twice with cold FACS buffer. The remaining cells were stained with (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.23.432366 doi: bioRxiv preprint CD45.2-APC (Clone 104; BioLegend) and 7AAD (Part 76332; Lot B226294 Biolegend) for 30 min at 4 °C, then washed and suspended in FACS buffer for flow cytometric sorting using FACS Aria II Cell Sorter (BD Biosciences). Sorted CD45.2 + 7AAD -cells with a purity greater than 95% and viability higher than 90% were used for single-cell RNA sequencing.
Tumor volumes were determined by caliper measurement using the formula V = (length × width 2 ) / 2. Freshly isolated tumor tissues were minced into approximately 1 mm 3 cubic pieces and digested using 0.1% collagenase IV (LS004186, Worthington), 0.002% DNAse I (D8071, Solarbio), and 0.01% hyaluronidase (H3506-1G, SIGMA), then incubated on a rocker at 37°C for 40-50 min. The digested cells were filtered through a 70 μm cell strainer and washed twice with cold FACS buffer. The remaining cells were stained with (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.23.432366 doi: bioRxiv preprint CD45.2-APC (Clone 104; BioLegend) and 7AAD (Part 76332; Lot B226294 Biolegend) for 30 min at 4 °C, then washed and suspended in FACS buffer for flow cytometric sorting using FACS Aria II Cell Sorter (BD Biosciences). Sorted CD45.2 + 7AAD -cells with a purity greater than 95% and viability higher than 90% were used for single-cell RNA sequencing.
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