At the start of each experiment, astrocytes were further purified by magnetic microbead-based negative selection to remove microglia using the CD11b MicroBead kit (Miltenyi Biotec, Auburn, CA; Catalog # 130-093-634). After removing the medium, cells were briefly washed with cold Tris-EDTA (pH 7.4, Sigma Aldrich, Saint Louis, MO; Catalog # 93302) and then enzymatically detached from T75 flasks using cold 0.25% Trypsin-EDTA (Mediatech, Inc., Manassas, VA; Catalog # 25-053-CI). Next, the cells were pelleted by centrifugation (300 × g, 5 min) and resuspended in MACS buffer (0.5% w/v bovine serum albumin [BSA] in PBS). Cells were manually counted using a hemocytometer and then washed and incubated with CD11b MicroBeads according to the manufacturer’s protocol. Cell separation was performed using LS Columns (Miltenyi Biotec; Catalog # 130-042-401) and a MidiMACS Separator (Miltenyi Biotec; Catalog # 130-042-302) outfitted with 30-µm pre-separation filters (Miltenyi Biotec; Catalog # 130-041-407).
30 m pre separation filter
Manufactured by Miltenyi Biotec
Sourced in Germany
The 30-µm pre-separation filter is a laboratory equipment designed to remove larger cells, cell clumps, and debris from cell suspensions prior to further processing. It is made of a polyethersulfone (PES) membrane with a pore size of 30 micrometers, which allows the passage of single cells while retaining larger particles.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b microbead kit, by Miltenyi Biotec (1 mentions)
Tris edta, by Merck Group (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Trypsin edta, by Corning (1 mentions)
Midimacs separator, by Miltenyi Biotec (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Liberase blendzyme 2, by Roche (1 mentions)
Dnase 1 grade 2, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Anti mouse cd16 cd32 fc block, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Accuri c6 plus flow cytometer, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
5 protocols using 30 m pre separation filter
1
Astrocyte Purification via Negative Selection
2
Isolation of Murine Splenocyte Subsets
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Single-cell suspensions from spleens of BALB/c and C57BL/6 mice were prepared via enzymatic digestion, as previously described [33 (link)]. For immunophenotyping, splenocyte suspensions were obtained using collagenase D digestion, whereas for the isolation of DC subsets, cell suspensions were prepared using liberase blendzyme II digestion followed by magnetic selection. Briefly, spleens were injected with Hanks’ balanced saline solution (HBSS) with Ca2+ and Mg2+ (Sigma-Aldrich, St. Louis, MO, USA) containing 100 U/mL of collagenase D or 100 Mandl U/mL of liberase blendzyme II and 20 µg/mL of DNAse I grade II (all from Roche Applied Science, Mannheim, Germany). Spleens were then mechanically disrupted with a needle and the obtained tissue fragments were vigorously pipetted and treated with 400 U/mL of collagenase or 400 Mandl U/mL of liberase solutions. After 60 min of incubation at 37 °C, tissue fragments were pipetted and strained through a nylon mesh to release single cells. Next, the cell suspensions were passed through 30 µm pre-separation filters (Miltenyi Biotec, Auburn, CA, USA), and after erythrocyte depletion with Red Blood Cell Lysis Buffer (Sigma-Aldrich), the splenocytes were counted using a Neubauer hemocytometer. Cell viability was assessed using a trypan blue exclusion assay.
3
Multiparametric Flow Cytometry Analysis of Islet Cells
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NPICCs were dissociated into single cells by treatment with TrypLE (Thermo Fisher Scientific, Germering, Germany) and washed with PBS + 10% foetal calf serum (FCS) and filtered through a 30-µm pre-separation filter (Miltenyi, Bergisch-Gladbach, Germany). Then cells were fixed/permeabilized using an intracellular staining buffer set and incubated with Fc-Block (anti-mouse CD16/CD32) for 10 min at room temperature (Thermo Fisher Scientific, Germering, Germany). Intracellular staining was performed using the following fluorochrome-labeled antibodies: mouse anti-insulin AF647 (clone T56-107), mouse anti-glucagon-PE (clone U16-850), mouse anti-somatostatin AF488 (clone U24-3545), mouse anti-Pdx-1-PE (clone 658A5), mouse anti-Pdx-1-AF488 (clone 658A5), mouse anti-Nkx6.1-AF647 and mouse anti Nkx6.1-PE (clone R11-560) (all from BD Biosciences, Heidelberg, Germany). All antibodies were pretested for appropriate dilution and for specificity using isotype control antibodies. Antibodies were incubated at 4 °C for 30 min followed by two washing steps with a permeabilization buffer. Flow cytometry data were acquired on a flow cytometer (BD Accuri™ C6 Plus Flow Cytometer, BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo software version 10.4 (TreeStar, Ashland, USA).
4
Flow Cytometry Analysis of Parasite Invasion
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Parasites were added to a HFF monolayer and allowed to invade for 1 hr. Then, cre recombinase-mediated recombination was induced by addition of 50 nM rapamycin in DMSO for 4 hr before washout. 22 hr after infection, parasites were syringe-released from the host cell, pelleted and washed twice in PBS. Parasites were resuspended in 0.5 ml 3% formaldehyde and fixed for 10 min. The suspension was centrifuged and the pellet washed in PBS before resuspension in PBS. To remove debris, samples were passed through a 30 µm pre-separation filter (Miltenyi Biotec, Bergisch Gladbach, Germany). 20,000 events were recorded using a BD LSR II flow cytometer (BD Biosciences California, United States). Killer Red was excited by the 561 nm laser and detected by a 600 long pass filter and either a 582/15, 610/20 or 620/40 band pass filter. YFP was excited by the 488 nm laser and detected a 505 long pass filter and either a 525/50 or 530/30 band pass filter. For quantification, and elimination of debris, total number of Killer Red+ parasites was considered 100%. This was performed at day 0, 35 and 65 of the experiment. For each condition, three biological replicates were analysed. At least 10000 Killer Red+ events were counted for each individual time point.
5
Snail Hemocyte Isolation and Profiling
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Pooled hemolymph (500µl) from 50 snails was recovered into 2ml tubes and mixed with 1.5ml of modified anticoagulant solution (98mM NaOH, 186mM NaCl, 1.7mM EDTA, 1.7mM citric acid) (13 (link)). Sample was passed through a 30µm pre-separation filter (Miltenyi Biotec) to eliminate cell aggregates and obtain a suspension of unique cells. Then hemocytes were counted using a Malassez chamber and cell viability was measured with trypan blue exclusion technique. Samples were then spin-down (2700g, 5min, 4°C) to pellet the hemocytes and re-suspended in 50µl of anticoagulant solution [30% of BGE medium (33 (link))] and 70% anticoagulant modified solution). To obtain a concentration of 1000 cells per microliter. Samples were then processed by MGX platform (IGH Montpellier, France) for scRNA droplet isolation (Chromium, 10X genomics) and RNA sequencing.
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