To separate single-cell suspensions, the collected individual spleens were passed through a 40-μm cell strainer in the presence of complete Roswell Park Memorial Institute (cRPMI) (Gibco BRL, Grand Island, NY, USA) medium supplemented with 10 % fetal bovine serum (Gibco BRL) and 1 % antibiotic (penicillin/streptomycin, Gibco BRL) using a sterile 5-mL syringe, and the cRPMI medium was removed by centrifugation (1500 × g for 3 min at 4 °C). Following this, single-cell suspensions were washed with phosphate-buffered saline (PBS) and centrifuged at 1500 × g for 3 min at 4 °C. To remove red blood cells (RBCs), single cells of spleen were incubated with 500 μL of RBC lysis buffer (Sigma-Aldrich) for 5 min at 4 °C, and the same volume of cRPMI medium as RBC lysis buffer was added and centrifuged (1500 × g for 3 min at 4 °C). The collected single cells were resuspended in cRPMI medium and used to analyze immune cells.
Crpmi
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CRPMI is a laboratory instrument designed for the measurement of C-reactive protein (CRP) levels in biological samples. It provides quantitative analysis of CRP concentrations, which can be used as an indicator of inflammation or infection. The CRPMI utilizes an automated process to measure CRP levels accurately and reliably.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Recombinant human il 2, by Miltenyi Biotec (2 mentions)
Anti cd28, by Thermo Fisher Scientific (2 mentions)
Cd14 and cd15 positive selection kits cd14 cd15, by Miltenyi Biotec (2 mentions)
Nlvpmvatv peptide from cmv pp65, by ProImmune (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Brefeldin a bfa, by Merck Group (2 mentions)
Facsaria, by BD (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
Il 33, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
70 m nylon mesh, by Celltreat (1 mentions)
Dnase, by Merck Group (1 mentions)
Ack buffer, by Lonza (1 mentions)
13 protocols using crpmi
1
Single-cell Isolation from Mouse Spleen
2
ILC2 Activation Assay Protocol
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For ILC2s activation, PBMCs were cultured with 1 µg/ml recombinant Der p 1 protein (Prospec, Rehovot, Israel),100 U/mL IL-2 (Sigma-Aldrich, St Louis, MO, USA) and 50 ng/ml IL-33 (Prepro Tech, Rocky Hill, NJ, USA) in complete RPMI medium 1640 supplement with L-Glutamine (cRPMI, ThermoFisher, Waltham, MS, USA), 10% fetal bovine serum (Sigma-Aldrich), and 1% PenStrep (Sigma-Aldrich). Cells were then cultured at 37°C in humidified atmosphere in 5% CO2 for 3 days. After that, cells were stained with Fixable Viability Dye eFlour780 and antibodies as previously described. Mean fluorescence intensity of activation marker (CD69 expression).
3
Multiparametric Flow Cytometry of TILs
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Peripheral blood (PBL) samples were drawn on day 7 post-treatment; 25 µL of fresh heparinized blood was incubated with fluorescence-conjugated antibodies (online supplemental table 2 ) for 30 min at 4°C in the dark. Tumors were harvested 3 or 7 days post-treatment, cut into small fragments, and digested in 1 mg/mL collagenase and 20 mg/mL DNase (Sigma) in serum-free RPMI-1640 for 30 min at room temperature (RT). TIL were filtered through 70 µm nylon mesh (Cell Treat), washed with 10 mL cRPMI, and collected by centrifugation (1500 rpm, 4 min). Pelleted cells were resuspended for staining and analysis by flow cytometry (see below). Lymph nodes (LNs) were harvested 7 days post-treatment and processed to obtain single-cell suspensions. Red blood cells were lysed with ACK buffer (Lonza) for 2 min at RT. Cells were then rinsed with cRPMI (ThermoFisher) and resuspended for antibody staining.
4
PBMC Isolation from Heparinized Blood
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PBMCs were isolated using Pancoll (PanBiotech GmbH, Aidenbach, Germany) or Ficoll (GE Healthcare Sciences, Chicago, IL) by density centrifugation from heparinized blood. Cells were washed and resuspended in Roswell Park Memorial Institute 1640 medium (Life Technologies, Carlsbad, CA) containing 10% vol/vol heat‐inactivated fetal bovine serum (HI‐FBS, Sigma‐Aldrich, St. Louis, MO; or Hyclone, GE Healthcare Sciences), 100 U/mL penicillin/streptomycin, 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid, β‐mercaptoethanol, and essential and non‐essential amino acids (cRPMI; all Thermo Fisher Scientific, Waltham, MA).
5
Purification and Functional Evaluation of gMDSC
6
Purification and Functional Evaluation of gMDSC
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gMDSC were enriched or depleted using sequential magentic bead isolation: CD14 and CD15 positive selection kits (CD14−CD15+) (Miltenyi Biotec) as per the manufacturer’s instructions. In selected cases gMDSC were highly purified by flow cytometry on the basis of CD11b, CD14 and CD15 expression on a FACSAria (BD Bioscience). Where indicated PBMC or PBMC enriched/depleted of gMDSC were stimulated with either: 0.5 μg/ml of an HLA-A and HLA-B-restricted peptide pool spanning the immune-dominant proteins of CMV, EBV and influenza (CEF) (JPT Peptide Technologies), 1 μg/ml overlapping peptides (pool of 15-mer peptides overlapping by 10 residues) spanning the core of HBV genotype D (JPT Peptide Technologies), 1 μg/ml HLA-A2-restricted NLVPMVATV peptide from CMV pp65 (ProImmune) or 0.5 μg/ml plate-bound anti-CD3 and 0.5 μg/ml anti-CD28 (eBioscience). All cultures were carried out in the presence of 20 IU/ml recombinant human IL-2 (Miltenyi Biotec) in cRPMI (Life Technologies) for 5-7 days at 37 °C. Specific responses were detected by intracellular cytokine staining after re-stimulation on day 4/6 in the presence of 1 μg/ml brefeldin-A (BFA, Sigma-Aldrich). Where indicated, 0.5 mM N-hydroxyl-nor-L-arginine (nor-NOHA) (Calbiochem), an arginase I specific inhibitor, was added to the culture on day 0.
7
THP-1 Macrophage Differentiation and TLR4 Expression
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The THP-1 (human monocytic cell line, American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 media (contained with 2 mM L-glutamine, 100 U of penicillin/mL, 100 mg of streptomycin/mL, 25 mM HEPES) (C-RPMI) and 5% fetal bovine serum, Gibco BRL, Gaithersburg, MD, USA). THP-1 cells (5 × 105 to 106 per mL) were seeded in serum-free medium (SFM, Gibco BRL) and treated with PMA (200 nM, 48 h) for differentiation [27 (link)]. After incubation, washed cells with C-RPMI three times to remove non-attached cells by aspiration, and the adherent cells were cultured with fresh IMDM complete medium (10% FBS, penicillin (100 units/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), sodium pyruvate (110 μg/mL) and GM-CSF (10 ng/mL, R&D Systems) for 2 days (37 °C, 5% CO2). To exclude the interference, the FBS was ultra-centrifuged and filtered (0.2 μM) prior to use to eliminate bovine serum exosomes. The well-differentiated macrophages were stained cellular plasma with CM-Dil and observed the morphology by fluorescence microscopy (Figure S2A ). The TLR4 expression pattern of THP-1 (unstimulated) and macrophages were determined by immunofluorescence staining by anti-TLR4 antibody. The TLR4 expression in membrane was similar between THP-1 and differentiated macrophages (Figure S2B ).
8
Maintaining THP-1-ASC-GFP Cell Line
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The THP-1-ASC-GFP (InvivoGen) cell line was maintained in cRPMI (GIBCO) supplemented with 100 µg mL–1 Zeocin (InvivoGen), at 37 °C and 5% CO2. Cells were used before reaching 20 passages.
9
PBMC Thawing and Cell Counting
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Upon testing, PBMCs were thawed as previously described [19 (link)]. Briefly, cryovials containing the PBMCs were subjected to a controlled increase in temperature in a water bath until completely thawed. Subsequently, the cell suspension was diluted using cRPMI (Bio-Whittaker, Walkersville, MA, USA) solution. The cell count and vitality were then determined under a UV microscope with Acridine Orange/Ethidium Bromide (Sigma–Aldrich, St. Louis, MO, USA/Fisher Scientific, Pittsburgh, PA, USA) staining. Before plating the PBMCs for further experimentation, the samples’ PBMC concentration was adjusted to 4 million cells per milliliter by adding cRPMI supplemented with fresh Glutamine (Gibco BRL, Grand Island, NY, USA), according to the previously counted cell concentration. This ensured uniformity in cell density and facilitated accurate and standardized analysis in subsequent assays.
10
Isolation and Culture of PBMCs
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Blood from donors was collected and processed within 2 h. PBMCs were separated using Histopaque 1077 at room temperature (Sigma-Aldrich) and following the protocol for mononuclear cell separation of SepMate-50 tubes (StemCells). Cells were resuspended in complete RPMI-1640 medium (10% Fetal bovine serum, 1% sodium pyruvate, 1% HEPES and 1% l -glutamine; cRPMI) (GIBCO), counted and plated in 24-well plates at a concentration of 0.5 × 106 cell mL–1 in 500 μl of cRPMI.
CD14 + cells were isolated from cryopreserved PBMCs. PBMCs were magnetic labeled with CD14 MicroBeads (Miltenyi Biotec) and isolated through positive selection using MACS Separation Columns MS (Miltenyi Biotec), according to the manufacturer’s protocol. Purified cells (~95% pure) were counted and plated in 96 well plates at a concentration of 0.5 × 106 cell mL–1 in 200 μl of cRPMI (GIBCO).
CD14 + cells were isolated from cryopreserved PBMCs. PBMCs were magnetic labeled with CD14 MicroBeads (Miltenyi Biotec) and isolated through positive selection using MACS Separation Columns MS (Miltenyi Biotec), according to the manufacturer’s protocol. Purified cells (~95% pure) were counted and plated in 96 well plates at a concentration of 0.5 × 106 cell mL–1 in 200 μl of cRPMI (GIBCO).
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