At necropsy, the stromal vascular fraction (SVF) was immediately isolated from epididymal fat by digesting with collagenase type II. Briefly, around 1g adipose tissues were extensively rinsed in PBS, minced and then digested with 1 mg/mL collagenase type II from Clostridium histolyticum (Sigma, St. Louis, MO) in DMEM with 5% FCS at 37 °C water bath with a shaking speed of 140 rpm for 30 min. The mix was then filtered through a 100 μm nylon cell strainer (BD Biosciences, San Jose, CA), followed by centrifugation at 300 × g for 5 min. The cells were then lysed with 1× RBC lysing buffer (Sigma, St. Louis, MO) for 5 min to get rid of contaminating red blood cells. After washing with PBS, the resulting pellets (SVF) were re-suspended with PBS and analyzed with flow cytometry.
Rbc lysing buffer
Manufactured by Merck Group
Sourced in United States, Macao
RBC lysing buffer is a laboratory reagent designed to selectively lyse red blood cells (RBCs) in a biological sample. It functions by disrupting the RBC cell membranes, releasing the hemoglobin and other cellular contents. This process enables the isolation and analysis of other cell types, such as white blood cells, from the sample.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Merck Group (4 mentions)
Facscalibur, by BD (4 mentions)
40 m cell strainer, by Greiner (3 mentions)
Glutamax and hepes, by Thermo Fisher Scientific (3 mentions)
Quadromacs separator magnet, by Miltenyi Biotec (3 mentions)
Ficoll paque premium, by GE Healthcare (3 mentions)
Anti cd14 magnetic bead, by Miltenyi Biotec (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
100 μm nylon cell strainer, by BD (2 mentions)
Type 2 collagenase from clostridium histolyticum, by Merck Group (2 mentions)
Dnase 1, by Worthington (2 mentions)
Macs bsa, by Miltenyi Biotec (2 mentions)
F7524, by Merck Group (2 mentions)
Muse cell analyzer, by Merck Group (2 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions)
Collagenase d, by Roche (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Gibco glutamax, by Thermo Fisher Scientific (2 mentions)
61 protocols using rbc lysing buffer
1
Isolation of Stromal Vascular Fraction from Adipose Tissue
2
Isolation of Stromal Vascular Fraction from Adipose Tissue
3
Isolation of Bone Marrow Adipocytes
4
Multiparametric Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometry of tissue culture cells, cells were isolated from mouse spleens and mechanically dissociated with a 40 µm cell strainer (Greiner Bio-One). Red blood cells were lysed with RBC lysing buffer (Sigma). Cells were labeled with aqua live/dead fixable viability dye in PBS (1:500), followed by surface antibodies (1:100) in staining buffer (1% BSA, 1% rat serum in PBS). Antibodies used for surface staining: PE/Dazzle anti-CD4 (RM4-5, Biolegend), APC/CY7 anti-CD8 (YTS156.7.7, Biolegend), PE/CY7 anti-CD44 (IM7, Biolegend), anti-CD69 (H1.2F3, BD Bioscience). For intracellular staining, cells were stained with aqua live/dead dye, followed by surface staining, fixation with perm/fixation solution (Thermofisher), and stained with intracellular antibodies against FITC anti-IFNγ (XMG1.2, Invitrogen) and AF647 anti-tumor necrosis factor α (TNFα) (MP6-XT22, Biolegend) in 1 x perm/wash buffer. Cells were washed and analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software.
5
Comprehensive Lung Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BAL and lung cells were treated with RBC lysing buffer (Sigma). Lungs were dissociated using a Lung Dissociation Kit (Miltenyi Biotec) and Gentle MACS (Miltenyi Biotec). LIVE/DEAD cells were stained with Fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific), and after Fc block with the 2.4G2 mAb (eBioscience), cells were stained with the following Abs: DR3-PE (4C12; BioLegend), SiglecF (E50-2440; BD Biosciences), CD11b (M1/70; BD Biosciences), CD11c (HL3; BD Biosciences), Ly6G (1A8; BioLegend), ST2-PE (101001PE, MD), CD90.2 (30-H12; BioLegend). For lineage markers for ILC2 staining, the following Abs were used: CD3-FITC (145-2C11; eBioscience), CD4-FITC (GK1.5; eBioscience), CD8- FITC (5H-10-1; BioLegend), CD19-FITC (1D3; BioLegend), NK1.1-FITC (PK136; BioLegend), CD11b-FITC (M1/70; BioLegend), CD11c-FITC (HL3; BD Biosciences), and Gr1-FITC (RB6-8C5; BioLegend). Flow cytometry analysis was performed on a Fortessa (BD Biosciences), and data were analyzed using FlowJo Software (version 10; FlowJo, Ashland, OR). Live CD45+ lung immune cells were separated into T cells (CD3+, CD90.2+), macrophages (CD11b+, CD11c+ SiglecF+), DCs (CD11c+ MHC class II+), neutrophils (GR1+, CD11b+ SigF-), eosinophils (Ly6C+, SigF+, CD11c-) and ILC2 (Lin- Thy1.2+ ST2+Sca1+).
6
Generating Murine Dendritic Cells from Bone Marrow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dendritic cells were generated from murine bone marrow cells by Inaba’s protocol23 (link) with minor modifications. Briefly, bone marrow cells were flushed from the femurs of mice, followed by incubation with red blood cell (RBC) lysing buffer (Sigma-Aldrich, St Louis, Missouri) to deplete the RBCs. The cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 8 ng/mL recombinant murine (rm) GM-CSF (PeproTech, Rocky Hill, New Jersey), and 2 ng/mL rmIL-4 (PeproTech). On day 1 of culturing, nonadherent cells were gently removed by gently shaking the dishes, and fresh medium was added. On day 3 of culturing, the LLC1 cell lysate prepared by 5 cycles of freezing and thawing of LLC1 cells were added into cultures, which contained 1 × 106 DCs, to obtain a final concentration of 5 mg/mL protein according to the study by Thumann et al.24 (link) On day 5 of culturing, the cultures were supplemented with 5 ng/mL recombinant human tumor necrosis factor-α (rmTNF-α) (PeproTech) to induce the maturation of the DCs, which were subsequently exposed to X-ray radiation at a dose of 0.2 Gy the next day. The X-rays were given using a precision linear accelerator (Elekta, Stockholm, Sweden) with a dose rate of 442.89 cGy/min.
7
Isolation and Cryopreservation of Mouse Bone Marrow-Derived Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs and BMMs were harvested from the femurs and tibias of age-matched (6–8 weeks) CO2-euthanized female BALB/c mice or male and female C57BL/6 J mice. BMDM media was supplemented with 10% M-CSF for differentiation, cells were seeded at 5×106 in petri dishes and cultured for 6 days, collected with cold PBS, and frozen in 90% FBS and 10% dimethyl sulfoxide (DMSO) in liquid nitrogen for later use. BMMs were isolated from BMDM fraction using EasySep Mouse Monocyte Isolation Kit (STEMCELL). Spleens were harvested from age-matched (6–8 weeks) CO2-euthanized female BALB/c mice, tissue was disrupted using the end of a syringe plunger on a 70-μm cell strainer and rinsed with FACS buffer (PBS + 2 mM EDTA). Cells were subjected to red blood cell lysis with RBC lysing buffer (Sigma) followed by neutralization in FACS buffer.
8
Immune Cell Isolation from Murine Spleen and Lung
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 3 or 10 wk post-immunization, animals were euthanized, and spleens were isolated by gross dissection. Tissues were processed into single-cell suspensions in complete medium (CM) consisting of RPMI 1640 supplemented with 20 mM L-glutamine, 10 mM HEPES, 50 ug/mL streptomycin, 50 U/mL penicillin, 50 mM 2-mercaptoethanol (Sigma–Aldrich, St. Louis, Missouri) and 10% FCS (Hyclone, Thermo Fisher Scientific, Waltham, Massachusetts). All reagents were purchased from Gibco unless otherwise specified. Red blood cell lysis was performed using RBC Lysing Buffer (Sigma–Aldrich). Lung tissues were also harvested, minced in sterile 25ml beakers, and incubated in complete digestion medium (CDM) in a 37° C water bath prior to processing into single-cell suspensions as described above. CDM consisted of CM without FCS, and with addition of 1mg/ml collagenase I and 30ug/ml DNase I (Worthington Biochemical Corp, Lakewood, NJ).
9
Assessing Host Cell Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess host cell depletion, 200μl of clodronate containing liposomes (www.clodronateliposomes.com ) was administered to mice by a single i.v. injection (via a lateral tail vein using 26G needles), or control untreated, 3 days prior to assessment by flow cytometry. For flow cytometry analysis, spleens were collected, chopped, collagenase/DNase treated (0.1% (1mg/ml) Collagenase D and 0.05% (0.5mg/ml) DNase in RPMI 1640 at room temperature for 30 minutes with constant mixing, then homogenized through a 100μm cell strainer to create a single cell suspension. RBC were lysed by incubating samples with RBC lysing buffer (Sigma-Aldrich) for 5 minutes at RT and splenocytes were washed in FACS buffer. Cells were assessed for viability by staining with LIVE/DEAD Fixable Aqua Dead Cell stain kit (Life Technologies), according to the manufacturers’ protocol. Cells were FcγR-blocked (αCD16/32 (clone 93)), and stained for surface markers by incubating with the following antibodies on ice for 20 minutes: B220-PB (RA3-6B2), Ly6G-APCCy7 (1A8), MHC-II-PE (M5/114.15.2), CD11b-PercpCy5.5 (M1/70), CD11c-APC (N418), Ly6c-FITC (HK1.4), F4/80-PeCy7 (BM8). Samples were fixed in 2% paraformaldehyde and acquired on a BD LSR Fortessa Cell Analyser (BD Biosciences). Data were analysed using FlowJo software (Tree Star).
10
Biological Sample Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were incubated for 16 h at 4°C, centrifuged for 10 min 10,000 rpm (9,877×g) to obtain sera, and stored at −80°C until analysis. Samples of vaginal secretions were obtained by washing the vaginal cavity with 250 µL of PBS containing 5% FBS and a cocktail of protease inhibitors (Roche, Mannheim, Germany), and then incubated on ice for 1 h. Vaginal secretion samples were centrifuged at 10,000 rpm (9,877×g) in an Eppendorf microfuge for 10 min at 4°C. They were then treated with 5 µL of 1 M PMSF and 5 µL of 1% NaN3, and then stored at −20°C until analysis. Splenocytes were purified from spleens squeezed on filters (Cell Strainer, 70 µm, Nylon, Becton Dickinson, Milan, Italy). Following red blood cell (RBC) lysis with RBC lysing buffer (Sigma-Aldrich), cells were washed with RPMI 1640 (Invitrogen) containing 10% FBS (Hyclone, Invitrogen), spun for 10 min at 1,700 rpm (557×g) in a benchtop centrifuge, and cultured in complete RPMI 1640 medium.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!