Mice were sacrificed under anesthesia. For isolation of stromal vascular cells, epididymal adipose tissues were removed, minced into small pieces and then incubated in a rotational shaker (200 rpm) at 37°C for 20 min in collagenase solution (1 mg/ml collagenase type 1, Worthington, UK, with 0.5% BSA and 10 mM CaCl2 in PBS). The digested tissue was passed through a 100-µm mesh and centrifuged (500 g, 10 min). The pellet containing the stromal vascular fraction was resuspended with erythrocyte lysis buffer (Sigma Aldrich, Germany) and centrifuged (500 g, 5 min). Cells were washed again in 2% FCS in PBS, resuspended in 250 µl 2% FBS in PBS, and counted via trypan blue exclusion. Livers were perfused with PBS and minced. Liver mononuclear cells were obtained by collagenase digestion of liver tissue for 30 min at 37°C on a rotation shaker (200 rpm). Following digestion, the cells were centrifuged (30 g, 1 min, RT) to pellet hepatocytes. Cells in the supernatant were then centrifuged at (310 g, 4 min, 4°C) and resuspended in 30% Percoll solution in HBSS followed by centrifugation (800 g, 30 min, RT) to enrich liver mononuclear cells. Erythrocytes were lysed in 8 ml erythrocyte lysis buffer (Sigma Aldrich, Germany) and 2 ml HBSS and centrifuged (300 g, 10 min, 4°C). Cell viability was checked with trypan blue exclusion.
Erythrocyte lysis buffer
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Erythrocyte lysis buffer is a solution used to lyse or break down red blood cells (erythrocytes) in a sample. Its core function is to facilitate the release of cellular contents, including nucleic acids and proteins, from erythrocytes. This buffer is commonly used in various biological and biochemical applications that require the isolation and analysis of cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1, by Worthington (7 mentions)
Collagenase d, by Roche (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Liberase tl, by Roche (2 mentions)
Dmem 10 fbs, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Worthington (2 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Trypan blue staining, by Bio-Rad (1 mentions)
μm cell strainers, by BD (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Type 2 collagenase solution, by Merck Group (1 mentions)
Phosphate buffer saline (pbs), by Merck Group (1 mentions)
40 μm nylon strainer, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alizarin red s, by Thermo Fisher Scientific (1 mentions)
Hla dr pe, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by BD (1 mentions)
Cd73 fitc, by Thermo Fisher Scientific (1 mentions)
32 protocols using erythrocyte lysis buffer
1
Isolation of Stromal Vascular and Liver Mononuclear Cells
2
Lung Water Content and BALF Analysis
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Lungs were collected after the mice were sacrificed following WBH. The wet weights of the organs were measured, and their dry weights were determined after the tissues were fully dried in an oven at 105°C. The water content was calculated as a percentage according to the following formula: 100×(wet weight-dry weight)/wet weight. One BALF aliquot was used immediately to measure the total cell counts. Erythrocytes were lysed using erythrocyte lysis buffer (Sigma, 1814389001), and the BALF was centrifuged at 400 g for 5 min and the supernatant was discarded. The pelleted cells were resuspended in 1.0 ml of PBS for the total leukocyte count by using a hemocytometer as previously described (44 (link)). The protein concentration in the supernatant was determined using bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA).
3
Isolation and Characterization of Visceral WAT
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Visceral white adipose tissue (WAT) was obtained from epididymal deposit of both WT and Elovl2−/− mice. The tissue was minced and digested with collagenase (250 U/ml in PBS, 2% bovine serum albumin (BSA), pH 7.4, v/v) for 30 min at 37 °C and the cell suspension was filtered through a 250-mm filter. Cells from the stroma-vascular fraction (SVF) were obtained after centrifugation at 300×g (room temperature) and treatment with erythrocyte lysis buffer (Sigma Aldrich) for 5 min. Finally, matrix fragments were removed using successive filtrations through 70 and 40-mm nylon meshes. Cells from SVF were then analyzed for cell surface markers by flow cytometry.
4
Isolation and Identification of FAP Cells
5
Isolation of Stromal Vascular Fraction from Adipose Tissue
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Fresh AT specimens (LPA and MAT) were dissociated enzymatically to obtain SVF. Briefly, samples were digested with type‐II collagenase (1 mg/ml collagenase in DMEM, both from Gibco, Thermofisher Scientific, Waltham, MA) for 45 minutes at 37°C in a shaking water bath. Samples were then washed with 2% FCS/PBS (Sigma Aldrich, St Louis, MO) and filtered sequentially through 100‐ and 70‐μm cell strainers (BD Falcon, Corning, NY). After centrifugation, pellets were resuspended in erythrocyte lysis buffer (155 mM NH4Cl, 170 mM Tris, pH 7.65, all from Sigma‐Aldrich) for 15 minutes at room temperature. Cells were washed again with 2% FCS/PBS and filtered through 40‐μm cell strainers (BD Falcon) to obtain single cell suspensions. Viable cells were counted following trypan blue staining (BioRad, Hercules, CA) on a haemocytometer.
6
Lymph Node Single-Cell Isolation
7
Isolation of Adipose Stromal Cells from Epididymal Fat
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Epididymal adipose tissue was washed with 1 × phosphate buffer saline (Life Technologies Inc., Burlington, ON, Canada) and visible blood vessels were removed from the fat pads. The minced tissues were digested for 1 h at 37 °C with type II collagenase solution (1.2 mg ml−1; Sigma-Aldrich Canada Ltd) and 2% bovine serum albumin fraction V (Roche Applied Science, Laval, QC, Canada) in phosphate buffer saline (pH 7.4) in an orbital shaking oven. Cell suspensions were centrifuged in polyethylene centrifuge tubes (Dow Corning Corporation, Midland, MI, USA) for 5 min at 250 × g and the floating layer composed of adipocytes was discarded. The pellet was resuspended in 5 ml erythrocyte lysis buffer (8.26 g NH4Cl, 1 g KHCO3 and 0.037 g EDTA in 1 liter water, pH 7.3; Sigma-Aldrich Canada Ltd) at room temperature for 5 min, followed by the addition of phosphate buffer saline (pH 7.4) to stop the lysis reaction. Cells were initially filtered through 100-μm nylon strainer and, subsequently, a 40-μm nylon strainer (BD Canada, Mississauga, ON, Canada). Finally, cell suspensions were centrifuged at 350 × g for 5 min at 4 °C and the pellet was stored at −80 °C until RNA extraction.
8
Multilineage Differentiation Potential of MSCs
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Cell media MCDB201, SMEM, DMEM, and RPMI 1640 were obtained from Life Technologies, human antibodies CD14-FITC, CD29-FITC, CD34-FITC, CD44-FITC, CD45-FITC, and CD90-FITC were from GeneTex, CD73-FITC and HLA-DR-PE were from eBioscience, mouse anti-human IgG1-FITC, IgG2a-FITC, and IgG2a-PE were from GeneTex, anti-CD3 and anti-CD28 were from BD Biosciences, Hoechst 33342, Lysotracker Green DND-265, and 6-carboxyfluorescein N-succinimidyl ester (CFSE) were from Invitrogen, Alizarin Red S, Alcian Blue, Oil Red O, phosphate-buffered saline (PBS), human serum albumin (HSA), erythrocyte lysis buffer, and all other chemicals were from Sigma-Aldrich and used without further purification.
9
Tissue Dissociation and Single-Cell Isolation
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Tissues were dissected into cold HBSS supplemented with Mg2+ and Ca2+, finely chopped and incubated in 400 U/ml Collagenase D (Roche) in HBSS for 25 min at 37°C, 5% CO2. Collagenase was quenched on ice by addition of final 10% FCS. Single cell suspensions were extracted from connective tissue by taking up and resuspending the digests five times. Erythrocytes were lysed by incubation in erythrocyte lysis buffer (Sigma) for 7 min at RT.
10
Isolation of PBMCs and PMN from Blood
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Blood collected in BD Vacutainer® citrated tubes (approximately 15 mL) was mixed and incubated with dextran 3% for 45 min at room temperature (RT). The supernatant was placed over Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and centrifuged at 650× g for 25 min at RT. The resulting halo of peripheral blood mononuclear cells (PBMCs) was collected and centrifuged for 10 min at 650× g. The pellet of polymorphonuclear leukocytes (PMN) was incubated with a specific erythrocyte lysis buffer (Sigma-Aldrich, Inc., St. Louis, MO, USA) for 5 min. Finally, PBMCs and PMN pellets were washed twice in Hank’s Balanced Salt Solution (HBSS; Capricorn, Ebsdorfergrund, Germany) prior to the following experiments.
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