Samples were finely minced with a scalpel and digested with 0.25% Trypsin-EDTA (ThermoFisher #25200056) for 30 minutes at 37°C with occasional agitation. The digestion was terminated by additional of RPMI (ThermoFisher #R8758) supplemented with 10% Fetal Bovine Serum (ThermoFisher # 16000044) and the cell suspension was filtered with a 70 μm mesh. A cell pellet was collected following 5 minutes of centrifugation at 300 ⨯ g. Red blood cells were removed with Red Blood Cell lysis buffer (BioVision, #5830-100). The cells were resuspended in PBS + 0.04% BSA (Sigma-Aldrich, #SRE0036), counted and 3,000 − 5,000 cells were loaded into the chromium controller (10X Genomics) following the manufacturer's instructions and processed using version 3 of the 3' scRNA-seq protocol (Suppl. Table S1 ).
Red blood cell lysis buffer
Manufactured by Abcam
Sourced in United States
Red Blood Cell Lysis Buffer is a solution used to selectively lyse (break down) red blood cells, while leaving other cell types intact. It is commonly used in the preparation of cell samples for flow cytometry, immunoassays, and other cell-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Luna fl dual fluorescence cell counter, by BioCat (3 mentions)
Acridine orange propidium iodide cell viability kit, by Logos Biosystems (3 mentions)
Chromium controller, by 10x Genomics (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dispase, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sre0036, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
True nuclear transcription factor staining protocol, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
70 μm cell strainer, by Miltenyi Biotec (1 mentions)
Human whole skin dissociation kit, by Miltenyi Biotec (1 mentions)
70 μm mesh cell strainer, by Corning (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase type 3, by Worthington (1 mentions)
Deoxyribonuclease 1 dnase 1, by Merck Group (1 mentions)
Ep1584y, by Abcam (1 mentions)
Pck 26, by Abcam (1 mentions)
15 protocols using red blood cell lysis buffer
1
Dissociation and Single-Cell RNA Sequencing
2
Single-cell RNA-seq Analysis of Tissue Samples
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Samples were finely minced with a scalpel and digested with 0.25% Trypsin-EDTA (Thermo Fisher, #25200056) for 30 minutes at 37°C with occasional agitation. The digestion was terminated by additional RPMI (Thermo Fisher, #R8758) supplemented with 10% fetal bovine serum (Thermo Fisher, #16000044), and the cell suspension was filtered with a 70-μm mesh. A cell pellet was collected following 5 minutes of centrifugation at 300 × g. Red blood cells were removed with red blood cell lysis buffer (BioVision, #5830-100). The cells were resuspended in PBS + 0.04% BSA (Sigma-Aldrich, #SRE0036) and counted, and 3,000 to 5,000 cells were loaded into the chromium controller (10x Genomics) following the manufacturer's instructions and processed using version 3 of the 3′ scRNA-seq protocol (Supplementary Table S1).
3
Isolation and Analysis of Murine Skin Cells
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Pigmented 7–10-wk-old mT/mG mice were shaved and their skin was depilated 48 h before sacrifice. The animals were killed by CO2 asphyxiation followed by cervical dislocation, and then the dorsal skin was removed. It was processed into a single-cell suspension by digestion with collagenase (Sigma-Aldrich), dispase (Sigma-Aldrich), and trypsin (Thermo Fisher Scientific) as in reference 44 (link). Briefly, the skin was minced, washed with PBS, and incubated for 12–13 h in a collagenase/dispase solution. The cells were further incubated for 5 min with trypsin, followed by mechanical disruption using 18-, 20-, and 22-gage needles. The cells were incubated for 5 min with red blood cell lysis buffer (BioVision) and then washed and flow-sorted for RFP-positive (dermal) cells or GFP-positive (epidermal and hair follicle keratinocyte) cells. RNA was extracted from these cells and gene expression was assayed using an Applied Biosystems 7900HT Fast Real-Time PCR System. We measured expression of murine Rev3l (Ex26Fwd: 5′-GTG AAT GAT ACC AAG AAA TGG GG-3′; Ex27Rev: 5′-GTG AAT GAT ACC AAG AAA TGG GG-3′; Probe: FAM-MGB-5′-TAC TGA CAG TAT GTT TGT-3′), Trp53, Kitl, Pomc, Wnt1, Edn1, and Tgfbr1 (all reactions except Rev3l were premade assays from IDT). Relative expression was achieved by comparing the ΔCt values of each sample with the ΔCt from one BK5.Cre; Rev3l+/lox mouse sample.
4
Isolation and Analysis of Murine Skin Cells
5
Comprehensive Immune Cell Profiling
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The immune cell populations (CD3 + T cells, CD4 + T cells, CD8 + T cells, regulatory T cells [CD4 + CD25 + FoxP3 + Tregs], myeloid-derived suppressive cells [CD11b + Gr-1 + MDSCs]) in spleens and tumor tissues with different treatments were analyzed by flow cytometry. Cell suspensions from spleens or tumor tissues were filtered with a nylon net (90 × 90 mm; Dakewe Biotech, Shenzhen, China) and red blood cells were lysed with Red Blood Cell Lysis Buffer (Catalog #5831-100; Biovision, CA, USA). The immune cells from tumor tissues were separated with a kit (WBC1092Z; HaoYang Biological, Tianjin, China).
For membrane staining, cells were incubated with combinations of antibodies (CD3, CD4, CD8, CD11b, Gr-1 and CD25). For intracellular staining for the antibody FoxP3, cells were fixed and permeabilized immediately after cell surface staining in accordance with the True-Nuclear™ Transcription Factor Staining Protocol (Biolegend). All antibodies were purchased from Biolegend and flow data were collected on a Gallios flow cytometer (Beckman Coulter, CA, USA). The data were analyzed using FlowJo 6 software (Tree Star).
For membrane staining, cells were incubated with combinations of antibodies (CD3, CD4, CD8, CD11b, Gr-1 and CD25). For intracellular staining for the antibody FoxP3, cells were fixed and permeabilized immediately after cell surface staining in accordance with the True-Nuclear™ Transcription Factor Staining Protocol (Biolegend). All antibodies were purchased from Biolegend and flow data were collected on a Gallios flow cytometer (Beckman Coulter, CA, USA). The data were analyzed using FlowJo 6 software (Tree Star).
6
Isolation of Oral Mucosal Cells and PBMCs
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The oral mucosa samples collected were immediately processed. The tissue samples were minced and underwent enzymatic digestion using the human Whole Skin Dissociation Kit (Miltenyi Biotec; No. 130-101-540) at 37°C. The digested sample was then filtered through a 70 μm cell strainer (Miltenyi Biotec; No. 130-110-916) to remove large particles. After centrifugation, the supernatant was removed, and the remaining cell deposit was resuspended in Red Blood Cell Lysis Buffer (Abcam; ab204733) for 5 minutes to lyse the erythrocytes. The cell suspension was washed and resuspended in D-PBS (BBI Life Science; E607009-0500), and magnetic cell separation (MicroBeads, Buffers, Columns, Separators: Miltenyi Biotec) was used to sort live cells. Fresh blood samples were collected in EDTA tubes, diluted with D-PBS, and then transferred onto Ficoll-Paque isolation solution (GE Healthcare; 17-5442-02). Peripheral blood mononuclear cells (PBMCs) were slowly and carefully collected after density gradient centrifugation, and then added with Red Blood Cell Lysis Buffer for 5 min. Afterward, PBMCs were washed twice with D-PBS.
7
Adoptive Transfer of Splenocytes
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Splenocytes were prepared according to a previous study [20 (link)]. At D5, AcGP mice were euthanized by cervical dislocation, and the spleen was removed immediately. The spleen was placed in 3 mL of ice-cold RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 2% fetal bovine serum (FBS) and minced using the plunger of a 1 mL injection syringe. The suspension was filtered through a 70 μm mesh cell strainer (Corning, Glendale, AZ, USA) into a 50 mL tube and washed with 5 mL of ice-cold PBS containing 2% FBS. The single-cell suspension was centrifuged at 500× g for 5 min at 4 °C. After removal of the supernatant, the pellet was incubated in 3 mL of red blood cell lysis buffer (Abcam, San Diego, CA, USA) for 3 min at room temperature, and hemolysis was stopped by adding 5 mL of ice-cold PBS containing 2% FBS. Splenocytes were pelleted by centrifugation at 500× g for 5 min at 4 °C, washed with ice-cold PBS containing 2% FBS, and resuspended in 3 mL of the same buffer. The suspension was then filtered through a cell strainer. A 200 μL aliquot of the suspension containing 1 × 107 cells was injected intravenously into the tail vein of each naïve recipient mouse.
8
Isolation and Characterization of NSCLC Cells
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All tissue samples were procured by the Human Tissue Procurement Service shared resource at the Winship Cancer Institute of Emory University in accordance with the approved institutional review board protocol. Tissues were digested for 3 hours in digestion buffer [DMEM/F-12 supplemented with 10 mM Hepes, 2% bovine serum albumin, 1× ITS (insulin-transferrin-selenium), hydrocortisone (0.5 μg/ml), and 1× normocin] containing type 3 collagenase (2 mg/ml; Worthington), hyaluronidase (100 U/ml; Sigma) at 37°C until fully digested. Cells were pelleted for 5 min at 300g, resuspended in red blood cell lysis buffer (Abcam) to lyse red blood cells, and pelleted again. Cells were then resuspended in digestion buffer containing deoxyribonuclease 1 (DNase 1) (200μg/ml; Sigma) and incubated for 10 min at 37°C. After DNase digestion, cells were pelleted, resuspended in media, and plated. Cells were grown in modified M87 media containing 2% FBS (50 ). The presence of the NSCLC marker TTF1 (EP1584Y) (1:50, Abcam) and pan-cytokeratin (clone PCK-26) (1:300, Abcam) and the absence of the fibroblast marker S100A4 (EPR2761) (1:100, Abcam) were used to verify the purity of these lines (51 (link)).
9
Single-cell RNA sequencing of aortic cells
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The complete aorta was perfused with cold PBS, isolated, and dissected to eliminate the surrounding periaortic fat. After mincing with scissors, a single-cell suspension was obtained through enzymatic digestion with elastase (Worthington, Lakewood, NJ, USA) and liberase (Roche, Basel, Switzerland) at 37°C. The preparation was passed through a 70-μm filter and treated with a Red Blood Cell lysis buffer (Abcam, Waltham, MA, USA). Single cells were resuspended in PBS containing 0.04% bovine serum albumin, counted, and assessed for viability using Trypan Blue.
Single-cell RNA sequencing was performed using the 10X Genomics Chromium platform at the UCLA Technology Center for Genomics and Bioinformatics. Raw sequencing data were demultiplexed and aligned using the Cell Ranger software from 10X Genomics, and the gene expression count matrix from Cell Ranger was obtained for downstream analyses.
Single-cell RNA sequencing was performed using the 10X Genomics Chromium platform at the UCLA Technology Center for Genomics and Bioinformatics. Raw sequencing data were demultiplexed and aligned using the Cell Ranger software from 10X Genomics, and the gene expression count matrix from Cell Ranger was obtained for downstream analyses.
10
Blood PBMC Isolation and Protein Analysis
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