Mouse thymic, splenic and lymph node cells for in vitro studies were obtained by passing the isolated organs through 70 μm cell strainers. The red blood cells contaminating spleen samples were lysed with ammonium chloride buffer. T cells were purified by negative selection using magnetic cell sorting (Total T cell isolation kit, Miltenyi Biotec). Thymic subpopulations were isolated by FACS sorting as follows: all CD45+ and then DN (CD4-CD8-), iSP8 (CD4-CD8+TCRint), DP (CD4+CD8+), SP4 (CD4+CD8-), SP8 (CD4-CD8+TCRhi). Where needed, isolated cells were cultured in RPMI 1640 medium supplemented with foetal bovine serum (10% v/v) and penicillin/streptomycin. Hepatic leukocytes were separated by Percoll gradient centrifugation (GE Healthcare, Freiburg, Germany). For T cell stimulation, the isolated cells were plated on culture dishes pre-coated overnight with anti-CD3 (diluted 1–10 μg/ml in PBS) and soluble anti-CD28 was added to the culture media at concentration 1 μg/ml.
Percoll gradient centrifugation
Manufactured by GE Healthcare
Sourced in Germany, United Kingdom, United States
Percoll gradient centrifugation is a laboratory technique used for the separation and purification of cells, organelles, and other biological particles. It involves the use of a colloidal silica-based medium to create a density gradient, which allows for the separation of different components based on their specific densities. The core function of this method is to enable the isolation and enrichment of target samples from complex mixtures.
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Lab products found in correlation
Dnase 1, by Roche (7 mentions)
Dnase 1, by Merck Group (6 mentions)
Dispase, by Thermo Fisher Scientific (4 mentions)
Collagenase 3, by Worthington (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (3 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (3 mentions)
Collagenase 8, by Merck Group (3 mentions)
70 μm cell strainer, by BD (3 mentions)
Phosphate buffered saline (pbs), by Corning (3 mentions)
Liberase tm with dnase 1, by Thermo Fisher Scientific (2 mentions)
40 m cell strainer, by BD (2 mentions)
Szx12 dissecting scope, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Omega Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
70 um cell strainer, by BD (1 mentions)
Red blood cell lysis solution, by BioLegend (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Collagenase a, by Roche (1 mentions)
37 protocols using percoll gradient centrifugation
1
Isolation and Culture of Murine Immune Cells
2
Isolation of Intestinal Lamina Propria Lymphocytes
3
Isolation and Culture of Primary Tumor Cells
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For primary cell isolation, tumor specimens were isolated at the time of surgery with informed consent and were gently minced into small pieces, then digested with 6 mL PBS containing 50 µL 25 mg/mL collagenase IV (Invitrogen, 17104019) and 25 µL 10 mg/mL DNase I (Roche, 10104159001) for 1 hour at 37°C. Cell suspensions were filtered twice and centrifuged at 1500 rpm for 5 min. Tumor cells and TILs were enriched and collected separately after Percoll gradient centrifugation (GE healthcare, 17-0891-01) following the manufacturer’s protocol. Afterwards, primary tumor cells were cultured with dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco, 1715753) and antibiotic antimycotic (Gibco, 15240062) for 2 weeks, then used for the indicated experiments.
4
Isolation and Culture of Decidual Immune Cells
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The fetal and placental tissues were carefully removed from the uteri of the mice at day 10.5 of gestation and washed in PBS. Minced uteri were digested in RPMI 1640 medium supplemented with collagenase Type IV and DNase I for 30
min at 37°C with gentle agitation. The total suspension was filtrated and enriched by discontinuous Percoll gradient centrifugation (1.130 g/ml, 60%, 40%, 20%; GE Healthcare Life Sciences, Little Chalfont, UK). After
centrifugation, the cells between 60% and 40% Percoll (the densities were between 1.062 and 1.077 g/ml, respectively) were roughly separated decidual immune cells (DICs) mixed with a small portion of decidual stromal cells. These
cells were then cultured in DMEM/F12 (37°C, 5%CO2) for 4 h to remove adherent stromal cells. The DICs were collected from the suspension and characterized by flow cytometry using PE-conjugated anti-mouse CD45 antibody.
The spleen was aseptically excised and stored in RPMI 1640 medium. A single-cell suspension was produced using a 10-ml syringe plunger to pass splenic tissue through a 70-mm mesh strainer into fresh medium. The cells were cultured
in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U[l penicillin, and 100 mg/ml amphotericin B at 37°C in 5% CO2.
min at 37°C with gentle agitation. The total suspension was filtrated and enriched by discontinuous Percoll gradient centrifugation (1.130 g/ml, 60%, 40%, 20%; GE Healthcare Life Sciences, Little Chalfont, UK). After
centrifugation, the cells between 60% and 40% Percoll (the densities were between 1.062 and 1.077 g/ml, respectively) were roughly separated decidual immune cells (DICs) mixed with a small portion of decidual stromal cells. These
cells were then cultured in DMEM/F12 (37°C, 5%CO2) for 4 h to remove adherent stromal cells. The DICs were collected from the suspension and characterized by flow cytometry using PE-conjugated anti-mouse CD45 antibody.
The spleen was aseptically excised and stored in RPMI 1640 medium. A single-cell suspension was produced using a 10-ml syringe plunger to pass splenic tissue through a 70-mm mesh strainer into fresh medium. The cells were cultured
in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U[l penicillin, and 100 mg/ml amphotericin B at 37°C in 5% CO2.
5
Isolation and Analysis of Myeloid Cells
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Blood and spleen samples from RP mice or controls were lysed by red blood cell (RBC) lysis solution (Biolegend, San Diego, CA). Cells were then passed through 70 um cell strainer (BD Biosciences, Franklin Lakes, NJ) to prepare monolayer cell suspension. Prolapsed or normal rectal tissues were enzymatically digested with enzyme cocktail containing collagenase IV (Worthington Biochemical Corp, Lakewood, NJ), dispase (Gibco), and DNAse I (Roche). Digested cell suspensions were subjected to 40% versus 80% percoll gradient centrifugation (GE Healthcare, Pittsburgh, PA) to enrich leukocytes. To phenotypically analyze immature myeloid cells, cells from blood, spleen, or RP tissue were stained with the following antibodies: PEcy7-CD45 (clone 30-F11), APC-CD11b (clone M1/70), and Percp-Cy5.5-Gr1 (clone RB6-8C5). Multicolor flow cytometry was performed on LSRII flow cytometer (BD Biosciences). All flow antibodies were obtained from Biolegend. Data were analyzed and presented by using Flowjo 9 software (Tree Star, Ashland, OR).
6
Isolation of CNS-Infiltrating Immune Cells
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Before the CNS was taken, mice were intracardially perfused with 10 ml PBS. The CNS was minced with a razor blade, digested for 45 min at 37 °C in a shaking water bath (1 mg/ml Collagenase A, Roche; 0.1 mg/ml DNaseI, Merck Millipore; in RPMI-1640 medium, PAN Biotech) and subsequently homogenized through a 70 µm cell strainer. After washing with PBS, immune cells were isolated by percoll gradient centrifugation (30%/78% 1.13 g/ml, GE Healthcare) at 2500 rpm, 30 min, 4 °C, w/o brake, harvested from the interphase and washed twice with PBS.
7
Isolation of Intestinal Lymphocytes
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Hematopoietic cells were isolated from macroscopically healthy human colonic mucosa, CRC tissue, or murine colon. Human tissues were obtained freshly after surgical removal of tumors from patients diagnosed with CRC. After removal of the Peyer’s patches and the adventitial fat, the murine colon was cut longitudinally. Prepared samples were washed with phosphate-buffered saline (PBS). For isolation of intraepithelial lymphocytes (IELs), the intestinal tissue was incubated in Hank’s balanced salt solution containing 1 mmol/L dithioerythritol, followed by a dissociation step using 1.3 mmol/L EDTA for 20 minutes at 37°C, respectively. To isolate lamina propria lymphocytes, the tissue was further cut in small pieces and minced with a scalpel. The remaining tissue was incubated for 45 minutes at 37°C on a shaking incubator in Hank’s balanced salt solution (with Ca2+ and Mg2+) with collagenase (1 mg/mL) and DNase I (10 U/mL), and supernatant was collected. Leukocytes were further enriched by Percoll gradient centrifugation (GE Healthcare, Princeton, NJ). If not stated otherwise, IEL and lamina propria lymphocytes were collected and pooled.
8
Murine MDSC Isolation and Generation
9
Isolation of Colon and Small Intestine Lamina Propria Cells
10
In vitro culture of P. falciparum asexual and sexual stages
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P. falciparum strain NF54 (WT NF54) was used in this study. Asexual blood stage parasites and gametocytes of WT NF54, the two 7-Helix-1-KO lines 2E6 and 1D12, the complementation line 7-Helix-1-KO(+) and the 7-Helix-1-HA line (for the generation of the 7-Helix-1 mutant lines, see below) were cultivated in vitro in human blood group A+ erythrocytes as previously described [82 (link),83 (link)]. All parasite stages were maintained in RPMI1640/HEPES medium (Gibco) supplemented with 10% v/v heat inactivated human A+ serum, 50 μg/ml hypoxanthine (Sigma-Aldrich) and 10 μg/ml gentamicin (Gibco). For cultivation of 7-Helix-1-KO parasites, the selection drug blasticidin (InvivoGen) was added in a final concentration of 5.4 μM; for cultivation of 7-Helix-1-KO(+) and 7-Helix-1-HA parasites, the selection drug WR99210 (Jacobus Pharmaceutical Company) was added in a final concentration of 2.5 nM. All cultures were kept at 37°C in an atmosphere of 5% O2 and 5% CO2 in N2. Gametocytes were enriched via Percoll gradient centrifugation (GE Healthcare Life Sciences) as described previously [84 (link)]. Gametogenesis was induced by adding xanthurenic acid in a final concentration of 100 μM dissolved in 1% v/v 0.5 M NH4OH/ddH2O and incubation for 15 min at room temperature (RT).
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