Vessels were flushed with phosphate-buffered saline (PBS) through the left ventricle and out the right atrium and then dissected free of adventitial tissue, minced with scissors into 1.5-ml Eppendorfs, and incubated for 1 h at 37°C with gentle rotation (20 rpm) in balanced salt solution (BSS) media containing 150 U/ml collagenase type IV (Sigma-Aldrich C5138), 60 U/ml DNase I (Sigma-Aldrich), 1 µM MgCl 2 (Sigma Aldrich), 1 µM CaCl 2 (Sigma-Aldrich), and 5% fetal bovine serum (FBS). Digested tissues were crushed through 35-µm cell-strainer caps (BD Biosciences) and quenched with 5 ml of cold BSS + 10% FBS in roundbottom tubes. Supernatant was removed after a 5-min, 320-g centrifuge, and the cell pellet was resuspended and quantified using a Z1 particle counter (Beckman Coulter).
35 m cell strainer cap
Manufactured by BD
35-µm cell-strainer caps are a laboratory equipment product used to filter and isolate cells from a cell suspension. They feature a 35-micron mesh pore size designed to retain cells while allowing smaller particles and debris to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (2 mentions)
Collagenase type 4, by Merck Group (2 mentions)
Z1 particle counter, by Beckman Coulter (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Cacl2, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Anti mouse igg microbeads, by Miltenyi Biotec (2 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Macs ls separation column, by Miltenyi Biotec (1 mentions)
4 protocols using 35 m cell strainer cap
1
Isolation and Quantification of Vascular Cells
2
Isolation of Primary Vascular Cells
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Vessels were flushed with phosphate-buffered saline (PBS) through the left ventricle and out the right atrium and then dissected free of adventitial tissue, minced with scissors into 1.5 ml microcentrifuge tubes (Eppendorf), and incubated for 1 hour at 37°C with gentle rotation (20 rpm) in balanced salt solution (BSS) media containing 150 U/ml collagenase type IV (Sigma-Aldrich C5138), 60 U/ml DNase I (Sigma-Aldrich), 1 µM MgCl 2 (Sigma Aldrich), 1 µM CaCl 2 (Sigma-Aldrich), and 5% fetal bovine serum (FBS). Digested tissues were crushed through 35 µm cell-strainer caps (BD Biosciences) and quenched with 5 ml of cold BSS + 10% FBS in round-bottom tubes. Supernatant was removed after a 5 minute, 320 g centrifugation, and the cell pellet was resuspended and quantified using a Z1 particle counter (Beckman Coulter).
3
Isolation of CD8+ T Cells from PBMCs
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PBMCs were harvested from the co-cultures at various time-points according to experimental set-up and resuspended in DPBS (Fig. 1 ). Cells were filtered through a 35-µm cell strainer cap (BD Biosciences), washed (400×g, 5 min), and resuspended in MACs buffer (DPBS (GIBCO), 0.5% bovine serum albumin (Fisher Scientific, Santa Clara, CA), 2 mM EDTA (Sigma)). Cells were labeled for CD8 followed by anti-mouse IgG Microbeads (Miltenyi Biotec (San Diego, CA)) and run through a MACS MS separation column (Miltenyi Biotec) per manufacturer's recommendation to obtain a positively selected CD8+ T cell population.
4
Isolation of Equine CD4+ T-cells
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Peripheral blood mononuclear cells were isolated from equine whole blood as described above and resuspended in DPBS. Cells were filtered through a 35 µm cell strainer cap (BD Biosciences), washed (400x g, 5 minutes), and resuspended in MACs buffer (DPBS, 0.5% bovine serum albumin (Gibco), 2 mmol/L EDTA). Cells were labeled for CD4 followed by anti‐mouse IgG Microbeads (Miltenyi Biotec). Cells were run through a MACS LS separation column (Miltenyi Biotec) per manufacturer's recommendation, and purity of CD4+ T‐cells after this column was confirmed by flow cytometry to be ≥95%. Isolated CD4+ T‐cells were resuspended in media.
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