Human colorectal adenocarcinoma samples were obtained under IRB-approved guidelines and with informed patient consent at Tongji Hospital of Huazhong University of Science and Technology, Wuhan, China. Fresh specimens were minced into small pieces with scissors. Completely minced pieces were then incubated in serum free DMEM/F12 medium (Life technologies, NY, USA) containing 1.5mg/ml collagenase IV (Gibco, NY, USA), 20 ug/ml hyaluronidase (Sigma-Aldrich, MO, USA), 1% penicillin/streptomycin (Life technologies, NY, USA) at 37°C for 1 to 2 hours. The specimens were mechanically dissociated every 15 minutes by pipetting with a 15-ml pipette. At the end of dissociation, cells were filtered through a 40-μm nylon mesh, washed with PBS. Red blood cells were then eliminated using red blood cells lysis buffer (Biolegend, CA, USA). Single cells were washed with PBS twice and resuspended in PBS.
Red blood cells lysis buffer
Manufactured by BioLegend
Sourced in United States
Red blood cells lysis buffer is a solution designed to selectively lyse (break down) red blood cells in a sample, while leaving other cells intact. This allows for the isolation and analysis of non-red blood cell components, such as leukocytes (white blood cells), in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Anti cd62l mel 14, by BioLegend (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Canto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Easysep mouse cd11c positive selection kit, by STEMCELL (1 mentions)
Granulocyte macrophage colony stimulating factor, by BioLegend (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Tnf α, by BioLegend (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Anti pe magnetic microbeads, by Miltenyi Biotec (1 mentions)
Anti gr1 pe antibody, by Miltenyi Biotec (1 mentions)
Facscalibur, by BD (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
6 protocols using red blood cells lysis buffer
1
Isolation of Colorectal Adenocarcinoma Cells
2
Isolation and Analysis of Hepatic and Splenic Lymphocytes
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Lymphocytes from fresh livers and spleens were prepared essentially as previously reported.26 (link) Briefly, excised liver from recipient mice was passed gently through a 200 μm steel mesh using a sterile syringe plunger. The liver cell suspension was collected, washed in PBS, and centrifuged for 10 min at 500 × g. The pellet was resuspended in 40% Percoll (GE Healthcare). The cell suspension was gently overlaid onto 70% Percoll and centrifuged for 25 min at 750 × g. Mononuclear cells were collected from the interface. Splenic cells were obtained from homogenized splenic tissue and filtered through a sieve. Contaminating red blood cells were lysed using red blood cells lysis buffer (BioLegend) and washed twice in PBS. Single cell suspensions obtained from the spleen and liver were subjected to flow cytometry analysis to detect surface antigens. The antibodies used to identify specific antigens were anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-CD44 (IM7), and anti-CD62L (MEL-14; all from BioLegend). Stained samples were assessed on a BD Accuri C6 Flow Cytometer (Becton Dickinson) and data analyzed using FlowJo Software (Tree Star).
3
Circulating Tumor Cell Enumeration by FACS
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The filtrates were then prepared for flow cytometry analysis based on fluorescence-activated cell sorting (FACS). Red blood cells were lysed by adding 10 mL of red blood cells lysis buffer (Biolegend, San Diego, USA), and samples were centrifuged for 10 min at 400× g (centrifuge 5810R, Eppendorf). The supernatant was discarded, and the pellet was resuspended in 200 µL PBS. The cells were fixed with 200 µL of a 4% formalin solution. An amount of 25 µL of counting beads solution (concentration of 5.2 × 104 counting beads per 50 µL, CountBright Absolute, Life Technologies, Zug, Switzerland) was added to each sample before analysis using a flow cytometer (BD Canto II, BD Biosciences). The acquisition was carried out with the BD FACSDiva Software (BD Biosciences). Forward, side scatter area, and signal height were recorded. Circulating tumor cells were detected using the forward scatter area vs. the PE-A. Fluorescence-activated cell sorting data were processed using FlowJo V10.0.8, Ashland, OR, USA.
4
Bone Marrow-Derived Dendritic Cell Culture
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Bone marrow-derived DCs were generated from flushed BM suspension from freshly dissected femurs and tibias. Cells were centrifuged for 5 min at 400 × g, treated with Red Blood Cells lysis buffer (BioLegend, San Diego, CA, USA) for 5 min, washed with PBS, centrifuged again and then cultured for 6 days in supplemented medium (RPMI medium; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 20 ng/ml granulocyte-macrophage colony stimulating factor (BioLegend, San Diego, CA, USA), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin–streptomycin, and 0.1% β-mercaptoethanol (Invitrogen, Carlsbad, CA, USA). Sp-DCs were purified from freshly isolated spleen using the EasySep Mouse CD11c Positive Selection Kit (StemCell, Vancouver, BC, Canada) according to the manufacturer’s instructions and then cultured in supplemented RPMI medium. Freshly isolated Sp-DCs or BM-DCs at day 6 were treated with LPS 100 ng/mL (S. typhimurium, L6143 Sigma-Aldrich, St. Louis, MO, USA) or TNF-α (20 ng/ml, BioLegend, San Diego, CA, USA) for different times as specified in each figure. In some experiments, BM-DCs were treated with LPS (100 ng/ml) together with anti-TNF-α blocking antibody (1 µg/ml, BioLegend, San Diego, CA, USA, clone MP6-XT22).
5
Isolation and Characterization of Murine MDSCs
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BM cells were flushed from femurs with PBS containing 2% FBS (Hyclone, Logan, UT, USA). Single cell suspensions were prepared by using a 70 µm filter and then red blood cells were deleted by incubating with red blood cells lysis buffer (Biolegend, San Diego, CA). Cells were incubated with Gr1-PE and CD11b-FITC fluorescence conjugated antibodies (Biolegend, San Diego, CA) for 20 min at room temper. Analysis was performed on a flow cytometer BD FACSCalibur (Becton Dickinson).Data was analysed by using BD CellQuest Pro software. For BM derived MDSCs sorting, BM cells were labelled with anti-Gr1-PE antibody and followed by incubating with anti-PE magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and then MDSCs were isolated by running the cell samples on AutoMACS (Miltenyi Biotec).
6
Dendritic Cell Derivation from Murine Bone Marrow
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Dendritic cells were derived from bone marrow cells with established protocols. Briefly, bone marrow was harvested from femurs and tibiae of BALB/c mice, treated with red-blood cells lysis buffer (BioLegend) and plated in R10 medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 1xAntiAnti, 12.5 mM HEPES, 4 mM Ultraglutamine) supplemented with either 20 ng/mL murine GM-CSF (BioLegend) or 200 ng/mL murine Flt3L (eBiosciences). Cells grown in GM-CSF were plated in 100 mm non-adherent petri dishes at a density of 2.5 × 105 cells/mL in 10 mL, whereas cells grown in Flt3L in wells of 6-well-plates at a density of 1.5–2 × 106 cells/mL. After 3 days, 5 mL R10 supplemented with 20 ng/mL murine GM-CSF were added to cultures grown in GM-CSF. Cells grown in GM-CSF were activated overnight at day 6, with 1 μg/mL LPS (Sigma), whereas cells grown in Flt3L were activated overnight at day 9, with 1 μg/mL poly I:C (GE Healthcare). Non-adherent cells were harvested, resuspended in R10 containing 10% DMSO and cryopreserved in liquid nitrogen.
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