Inflamed mucosa (n = 3 AFRS nasal polyps, n = 1 mucosa adjacent to mycetoma) and control mucosa from inferior turbinates (n = 4) were each taken from the same patient and prepared as previously decribed.5 (link) Briefly, tissue was incubated for 5 hours at 37°C in Dulbecco’s Modified-Eagle Medium (Lonza, Basel, Switzerland) with 5μg/mL Brefeldin A (Sigma-Aldrich, St. Louis, MO). Tissue was enzymatically digested releasing single cells into suspension.5 (link) Ethylenediamine tetra-acetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO) at final concentration 2mM was added to quench the digestion. Debris was removed using a 40-μm cell strainer and red blood cells (RBCs) were removed using an RBC lysis buffer (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were serially washed in 2% BSA in PBS (Sigma-Aldrich, St. Louis, MO) for analysis by flow cytometry.
Rbc lysis buffer
Manufactured by Miltenyi Biotec
Sourced in United States
RBC lysis buffer is a reagent used to selectively lyse or break down red blood cells (RBCs) in a sample, leaving other cell types intact. It is a commonly used tool in cell isolation and purification protocols.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (3 mentions)
Dulbecco s modified eagle medium, by Lonza (3 mentions)
Dnase 1, by Roche (2 mentions)
Liberase tl, by Roche (2 mentions)
Liberase, by Merck Group (2 mentions)
Hepes buffer, by Thermo Fisher Scientific (2 mentions)
Cacl2, by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Countess 2 fl automated cell counter, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (1 mentions)
70 μm cell strainer, by BD (1 mentions)
13 protocols using rbc lysis buffer
1
Isolation of Immune Cells from Inflamed Nasal Mucosa
2
Bronchoalveolar Lavage Cell Processing
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The BAL sample was filtered through a 70μm cell strainer and then centrifuged at 1200rpm for 5 min. The supernatant was removed and stored at -80°C to be used as “BAL fluid” for cytokine measurements (below). The pellet was mixed with RBC Lysis buffer (Miltenyi), gently vortexed and incubated at room temperature (RT) for 15 minutes. The cells were then spun at 1200rpm for 5 min and supernatant was aspirated. Cell pellet was resuspended in 10mL phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS). Cells were counted and cytospin slides were made for counting cell types (see below). The remainder of the cells were centrifuged at 1200rpm for 5 min and supernatants was aspirated. The cells were resuspended in 7% dimethyl sulfoxide(DMSO)/FBS solution and transferred to 1mL cryovials (max 20x10^6 cells per vial). The cryovials were frozen overnight at -80°C with gradual cooling (1 degree C/min) and then placed into liquid nitrogen the following morning.
3
Dissociation of Tumor Tissue for Single-Cell Analysis
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Tumor tissue was dissociated within 2 h after collection. Each tissue was minced into ≤1–2 mm pieces in 1 mL enzyme mix (enzymes H, R, and A from Miltenyi Biotech in RPMI‐1640 medium). The minced tissue suspension and the remaining enzyme mix were transferred to a gentleMACS C tube and then placed onto the gentleMACS Octo dissociator (Miltenyi Biotech). The gentleMACS program 37C_h_TDK_3 was run for dissociation after attaching the heating elements. The homogenate was centrifuged briefly and filtered through a 70 μm cell strainer (BD Biosciences). The cell suspension was then centrifuged at 300 g for 7 min, the supernatant was aspirated, and the pellet was resuspended in RPMI‐1640 medium (Gibco). Red blood cells were lysed by incubating the pellet in RBC lysis buffer (Miltenyi Biotec) for 10 min at 4°C, after which 10 mL chilled RPMI‐1640 medium was added, centrifuged at 300 g for 10 min at 4°C, and the cell pellet was resuspended in RPMI‐1640. The number of cells and fractions of live cells were counted using Trypan blue and Live/Dead Viability/Cytotoxicity Kit (Thermo Fisher Scientific) in a Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Cell suspensions with 75% or more viable cells were then diluted to a final concentration of ~1000 cells/μL.
4
Single-cell RNA-seq of Synovial Tissue
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Synovial biopsy and tissue analysis was approved by the Kantonal Ethic Commission Zurich, Switzerland (refs: 2019-00115 and 2016-02014). All patients signed informed consent forms. Patient's characteristics were as follows:
One part of the synovial biopsies was embedded in paraffin and histologically analyzed as previously described (43 (link),44 (link)). For scRNA-seq synovial biopsies were mechanically minced and enzymatically digested with 100 ug/ml Liberase TL (Roche) and 100 ug/ml DNAseI (Roche) in pre-warmed RPMI 1640 containing 2 mM glutamine and 25 mM HEPESGibco for 30 min at 37°C. The digestion was stopped by adding 20% (v/v) fetal calf serum (FCS) and the dissociated tissue was filtered through a 40 um cell strainer. Red blood cells were lysed with RBC lysis buffer (Milteny Biotec). Cell viability was analyzed with a LUNA-FL dual fluorescence cell counter (88% and 90%).
One part of the synovial biopsies was embedded in paraffin and histologically analyzed as previously described (43 (link),44 (link)). For scRNA-seq synovial biopsies were mechanically minced and enzymatically digested with 100 ug/ml Liberase TL (Roche) and 100 ug/ml DNAseI (Roche) in pre-warmed RPMI 1640 containing 2 mM glutamine and 25 mM HEPESGibco for 30 min at 37°C. The digestion was stopped by adding 20% (v/v) fetal calf serum (FCS) and the dissociated tissue was filtered through a 40 um cell strainer. Red blood cells were lysed with RBC lysis buffer (Milteny Biotec). Cell viability was analyzed with a LUNA-FL dual fluorescence cell counter (88% and 90%).
5
Neutrophil Isolation from Mouse Blood
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A MACS system and a mouse neutrophil isolation kit (Miltenyi Biotec, Teterow, Germany) were used to isolate neutrophils by negative selection. Whole blood was collected from eight mice from the same group and lysed with red blood cell (RBC) lysis buffer (Miltenyi Biotec). The cells were resuspended in buffer and then incubated with a neutrophil biotin-antibody cocktail for 10 min at 4 °C. After washing once in buffer, the cell pellets were incubated with Anti-Biotin MicroBeads (Neutrophil Isolation Kit, Miltenyi Biotec) for 15 min at 4 °C. Then, the cell pellets were washed at 300 × g for 10 min, and the suspension was passed through an MS Column (Miltenyi Biotec). The unlabeled cells representing enriched neutrophils were collected.
6
Isolating Nasal Polyp Immune Cells
7
Single-Cell Tumor Dissociation Protocol
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Single-cell preparation for scRNA-seq and mass cytometry validation was performed according to a previous study [16 (link)]. In brief, tumors were cut into approximately 1 mm3 pieces and digested in Hank’s balanced salt solution (HBSS) containing 1.5 mg/ml collagenase IV (Sigma‒Aldrich), 1 mg/ml hyaluronidase (Sigma‒Aldrich) and 500 µg/ml DNase I (GoldBio) while rotating at 200 rpm and 37 °C for 30 min. Cell suspensions were subsequently passed through a 70-µm cell strainer (BD, Biosciences), followed by centrifugation at 400 × g for 10 min. Red blood cells were lysed with RBC lysis buffer (Miltenyi Biotec, 130-094-183) for 2 min on ice, and dead cells were removed with a dead cell removal kit (Miltenyi Biotec, 130-090-101) following the manufacturer’s instructions.
8
Isolation and Enumeration of Immune Cells
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Lungs and spleens were processed as described by Fox et al. [25 (link)]. Briefly, a portion of the left cranial lung lobe and spleen from each hamster were aseptically transferred from PBS into DMEM then teased apart. Lungs were treated with a solution of DNase IV (500 units/mL) and Liberase (0.5 mg/mL) for 30 min at 37 °C to dissociate and digest collagen. Both lung and spleen cells were homogenized using a syringe plunger and passed through a 70 µm filter to prepare single cell suspension. Erythrocytes were lysed using Gey’s RBC lysis buffer (0.15 M NH4Cl, 10 mM HCO3) and cells were resuspended in 1 mL of complete media.
For blood, buffy coat was harvested by adding equal volume of PBS + 2% FBS to the blood and centrifuging at 800× g for 10 min at 25 °C with brakes off. The buffy coat was collected and washed, and erythrocytes were lysed using 1× Miltenyi RBC lysis buffer (Miltenyi, CA, USA). Cells were washed and resuspended in 1 mL complete media. After adding absolute counting beads (Invitrogen), total cell numbers of lung, spleen and blood were determined by flow cytometry analysis using an LSR-II (BD).
For blood, buffy coat was harvested by adding equal volume of PBS + 2% FBS to the blood and centrifuging at 800× g for 10 min at 25 °C with brakes off. The buffy coat was collected and washed, and erythrocytes were lysed using 1× Miltenyi RBC lysis buffer (Miltenyi, CA, USA). Cells were washed and resuspended in 1 mL complete media. After adding absolute counting beads (Invitrogen), total cell numbers of lung, spleen and blood were determined by flow cytometry analysis using an LSR-II (BD).
9
Purification of Alveolar Epithelial Type 2 Cells
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P18 mouse lungs were collected from +/Y and Mecp2/Y mice at 9 a.m. A single-cell suspension was made as published, with modifications (56 (link)). Briefly, lungs were excised and incubated in fresh Hank’s balanced salt solution (HBSS) with 5 U/ml dispase, 0.1% collagenase I and 0.002% DNase I for 30 min at room temperature. The lung was then disintegrated using forceps in a 6 cm petri dish. The cell suspension was filtered through 100, 70 and 30 μm nylon cell strainers. Red blood cells were lysed using RBC lysis buffer (Miltenyi Biotec). Cells were pelleted and resuspended in 500 μl staining media [SM: HBSS, 2% fetal bovine serum (Wisent), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.2]. Cells were counted using the TC10/TC20 cell counter (BioRad). Approximately 3 million cells per sample were stained with fluorochrome-conjugated antibodies from BD Biosciences for 30 min on ice in the dark. Antibodies used were as follows: CD45.2 (558702), CD31 (561814), CD326 (563478), I-A/I-E (553623) and Podoplanin (566390). Cells were sorted using a MoFlo Astrios (Beckman Coulter) cell sorter. AE2 cells were as follows: CD45.2−, CD31−, CD326+, I-A/I-E+, Podoplanin−. Following sorting, cells were pelleted and re-suspended in fresh SM.
10
Isolation and Fixation of BALF Cells
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BALF samples were centrifuged at 500 × g for 5 min at 4 °C and added RBC lysis buffer (130-094-183, Miltenyi Biotec, Seoul, Korea) and incubated for 3 min at 37 °C. 1x PBS was added and centrifuged at 215 × g for 5 min at 4 °C and counted the number of cells. The additional centrifuge was performed at 215 × g for 5 min at 4 °C. Warmed fixation buffer (420801, BioLegend, CA, USA) was added and incubated for 10 min at room temperature in the dark. BAL cells were washed with FACS buffer (PBS 50 ml + FBS 1 ml + 0.5 M EDTA 20 μl) and an extra centrifugation step was carried out at 215 × g for 5 min at 4 °C.
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