Lung cells were dissociated as previously described (Sharma et al., 2022 (link)). The right lung was dissociated to a single cell suspension with Multi Tissue Dissociation Kit 2 (130–110–203, Miltenyi) according to the manufacturer’s protocol using the gentle MACS Dissociator (Miltenyi). The cell suspension was filtered through a 70 μm strainer, pelleted by centrifugation, and cleared of red blood cells with ACK Red Blood Cell Lysis buffer (BP10-548E, Lonza). Live cells were enumerated using trypan blue exclusion counting on a Countess Automated Cell Counter Hemocytometer C10227 (Invitrogen). Lung cells were then used as described below for Fluorescence-Activated Cell Sorting (FACS) analysis. Additionally, ten million lung cells per animal were used for CD45+ enrichment using the CD45 MicroBeads kit (cat# 130–109–682, Miltenyi) per manufacturer’s instructions.
Ack red blood cell lysis buffer
Manufactured by Lonza
Sourced in United States
ACK red blood cell lysis buffer is a solution used to remove red blood cells from a cell sample. It functions by causing the lysis, or rupturing, of red blood cell membranes, allowing for the isolation of other cell types for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Multi tissue dissociation kit 2, by Miltenyi Biotec (2 mentions)
Cd45 microbeads kit, by Miltenyi Biotec (2 mentions)
Macs dissociator, by Miltenyi Biotec (2 mentions)
Countess automated cell counter hemocytometer c10227, by Thermo Fisher Scientific (2 mentions)
70 m nylon strainer, by BD (2 mentions)
B 5 r lsr 2, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Diaclone (1 mentions)
Edta blood collection tubes, by BD (1 mentions)
Human fc blocking reagent, by Miltenyi Biotec (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
7 protocols using ack red blood cell lysis buffer
1
Lung Cell Dissociation and CD45+ Enrichment
2
Antigen-Specific Cytotoxicity Assay
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Splenocytes from female mice were pulsed with 1 µg/ml of the MHC-I-restricted OVA257-264 peptide or HIV-1 gag peptide as a control before labeling with 5 or 0.5 µM of CFSE (Invitrogen, Merelbeke, Belgium), respectively. Labeled cells were mixed at a 1:1 ratio and a total of 1.5 × 107 mixed cells were adoptively transferred into immunized mice 3 days after the second mRNA treatment. Forty eight hours later, splenocytes from mice were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA). Next, splenocytes were analyzed by flow cytometry. Percentage of antigen-specific killing was determined using the following formula: (1 − (%CFSEhi cells/%CFSElow cells)treated mice/(%CFSEhi cells/% CSFElow cells)non-treated mice) × 100. The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA) and analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on their SSC and FSC. Next CFSE-positive cells were selected. See Supplementary Fig. 5 for gating strategy.
3
Induction of Tumor-Specific T-Cell Responses
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C57BL/6 mice were inoculated with 5 × 105 B16 cells, and at days 6 and 10, the mice were treated with saline or 10 µg mRNA encoding Fluc, tBid, or MLKL. Two days after the second treatment, spleens were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA) and 2.5 × 105 cells were cultured for 24 h in wells of anti-IFN-γ (Diaclone, Besancon, France) pre-coated 96-well plates in the presence of 10 µg/ml peptide. The following synthetic, high-performance liquid chromatography-purified peptides were used for restimulation: OVA 257–264 (SIINFEKL), OVA 323–339 (SQAVHAAHAEINEAGR), CT26-M20 (PLLPFYPPDEALEIGLELNSSALPPTE), CT26-M26 (VILPQAPSGPSYATYLQPAQAQMLTPP), CT26-M03 (DKPLRRNNSYTSYIMAICGMPLDSFRA), CT26-M37 (EVIQTSKYYMRDVIAIESAWLLELAPH), CT26-M27 (EHIHRAGGLFVADAIQVGFGRIGKHFW), B16-M30 mut (PSKPSFQEFVDWENVSPELNSTDQPFL), and B16-M30 WT (PSKPSFQEFVDWEKVSPELNSTDQPFL).
4
Isolation and Immunophenotyping of Murine Myeloid Cells
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Peripheral blood (100 μl) from the retro-orbital sinus was collected into EDTA blood collection tubes (BD Biosciences San Jose, CA). Blood samples were mixed with 250 μl of 2% dextran (Sigma-Aldrich, St. Louis, MO) and incubated at 37°C for 30 min to sediment red blood cells. Mononuclear cells in the supernatant were transferred and centrifuged at 400g. Contaminating red blood cells were removed by the addition of Ammonium-Chloride-Potassium (ACK) red blood cell lysis buffer (Lonza, Alpharetta, GA). Samples were then blocked with 5% mouse Fc blocking reagent (2.4G2; BD Biosciences) and 5% human Fc blocking reagent (Miltenyi Biotec, Auburn, GA) in fluorescence-activated cell sorting (FACS) buffer [1% FBS and 1 mM EDTA in phosphate-buffered saline (PBS)] before antibody staining. Blocked cells were stained with antibodies against hCD8-allophycocyanin (APC; SK1), mLy-6C-phycoerythrin (PE; AL-21), mLy-6G-Fluorescein isothiocyanate (FITC; 1A8), mF4/80-PE-Cy7 (BM8), hCD4-APC/Cyanine7 (APC-Cy7; SK3), and hCD20–Violet 421 (2H7) at 4°C for 30 min. Labeled cells were washed and fixed in 1% paraformaldehyde (PFA).
5
Lung Cell Isolation and Characterization
6
Breast Cancer Pleural Effusion and Ascites Protocol
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Pleural effusion and ascites samples were obtained from breast cancer patients admitted to the Department of Gynecology, University Clinic Mannheim, University of Heidelberg, and the National Center for Tumor Diseases Heidelberg (NCT). The studies were approved by the ethical committee of the University of Mannheim (case number 2011‐380N‐MA) and the University of Heidelberg (case number S‐295/2009) and conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Written informed consent was obtained from all patients. Samples were processed as previously described (Gomis et al, 2006 ). Briefly, pleural effusion or ascites fluids were centrifuged at 300 g for 5 min. Cell pellets were lysed using ACK red blood cell lysis buffer (Lonza) according to manufacturer's instructions. Cells were washed with PBS (Sigma‐Aldrich) and plated for culture.
7
Immune Cell Profiling in Tumor Tissue
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The flow cytometric analysis was employed for the analysis of deletion of CD8+T, CD4+T, and NK cells, and the density of CD8+T in tumor tissue suspensions. For the analysis of deletion of CD8+T, CD4+T and NK cells, the following antibodies were used for staining cells: CD3-Violet 421 (145-2C11), CD4-allophycocyanin (APC) (GK1.5), CD8-PE-Cy7 (53-6.7), NK1.1-PerCP. For analyzing the density of CD8+T in tumor tissue suspensions, single-cell suspensions from tumors were prepared, followed by the cell lysis with ACK red blood cell lysis buffer (Lonza) and then antibody staining (CD3-Violet 421, CD4-APC, and CD8-PE-Cy7) at room temperature for 30 min. For analyzing the influence of Ad-hTERT on IL-10R expression on CD8+T cells, mouse peripheral CD8+T cells were enriched and incubated with Ad-hTERT (Ad-hTERT:CD8+T cells = 100:1) for 72 h, then stained with anti-IL-10R (1B1.3a), followed by incubation with rabbit anti-mouse IgG Fc secondary antibody-FITC (ThermoFisher Scientific). After labeling, the cells were fixed in 1% paraformaldehyde, and 10 (5 (link)) events were collected and analyzed on a CytoFLEX flow cytometer (Beckman Coulter). Analysis was carried out using the CytExpert software (Beckman Coulter).
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