Tumor cell media was collected and analysed for soluble TGFα, EGF, TNFα, HB-EGF, AREG, BTC and EPR using an ELISA kit (Neobioscience Technology Company) according to the manufacturer's instructions. For normalization, the cells were trypsinized and the live cell fraction was counted using a handheld automated cell counter (Millipore Corporation).
Handheld automated cell counter
Manufactured by Merck Group
Sourced in United States, Germany
The Handheld Automated Cell Counter is a compact, portable device designed for quick and accurate cell counting. It utilizes advanced optics and automated algorithms to provide reliable cell concentration and viability measurements. The core function of this equipment is to streamline the cell counting process, enabling efficient and consistent data collection.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Puromycin, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
96 well flat bottomed cell culture plates, by Corning (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Collagenase, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sodium oleate, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mab478, by R&D Systems (1 mentions)
Human mscs, by Cyagen (1 mentions)
Huxma 90011, by Cyagen (1 mentions)
Dl β hydroxybutyric acid sodium salt, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by PAN Biotech (1 mentions)
Cc 2509, by Lonza (1 mentions)
Glomax luminometer, by Promega (1 mentions)
19 protocols using handheld automated cell counter
1
Quantification of Tumor Cell Secretome
2
PBMC Isolation and Stimulation Protocol
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Whole heparinized peripheral blood was diluted 1:2 (vol/vol) in an RPMI 1640 medium (Sigma Aldrich, Darmstadt, Germany). Peripheral blood mononuclear cells (PBMC) were separated on a Ficoll (Eppendorf, Hamburgo, Germany) density gradient by centrifugation at 300 g for 30 min at room temperature. Then, the cells in the interface were collected, washed twice, and counted using a handheld automated cell counter (Millipore Co., MA, USA). The viability was assessed by trypan blue dye exclusion. The PBMC were cultured in 96-well flat-bottomed cell culture plates (Costar, MA, USA) at 2 × 105 cells/well in an RPMI-1640 medium supplemented with one mM sodium pyruvate and two mM L-glutamines and incubated at 37 °C in a 5% CO2 humidified chamber. After 24 h, the culture medium was removed, and a fresh culture medium supplemented with 10% heat-inactivated fetal calf serum and/or Der p (0.5 μg/mL), LPS (10 μg/mL), or α-MSH (1 μg/mL) was added. The Con A mitogen (1 μg/mL) was used as a cell stimulation positive control (Table S2 ). After 72 h of culturing, the cells were harvested and processed to measure the intracellular FOXP3 expression and the expression of CD24, CD69, and TLR4 on the cell surface by flow cytometry. The supernatants were collected and stored at −70 °C to determine soluble cytokines.
3
PBMC Isolation from Heparinized Blood
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Blood samples were collected by venipuncture. Heparinized peripheral blood was diluted 1:2 in PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2P04, pH 7.4). Peripheral blood mononuclear cells (PBMCs) were separated on a Ficoll density gradient with centrifugation at 500 ×g for 30 min at room temperature. Then, cells in the interface were collected, washed twice, and counted using a handheld automated cell counter (Millipore Co., Billerica, MA); viability was assessed with eosin dye exclusion.
4
Cytotoxicity Assay of DOX-Loaded Liposomes
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Monolayer HeLa cells at ~85% confluence were suspended by trypsinization, and the cell density was determined with a Handheld Automated Cell Counter (Millipore, Burlington, MA, USA). The cells were then diluted to ~80,000 cells/mL in complete growth media and seeded into 96-well Clear Microplates (Corning, NY, USA) at ~8000 cells/well by transferring 100 μL of the cell suspension into each well. The cytotoxicity assay was carried out on the cells 8 h after they were seeded. The cells were washed with PBS and treated with DOX-loaded liposomes or free DOX solutions in complete growth media at incremental concentrations. The pH of the media (10 mL) was adjusted to 7.4, 7.0, 6.5, and 6.0 with glacial acetic acid. After 12-h incubation, the media was removed, and the cells were washed with 100 μL PBS buffer and supplemented with 100 μL/well complete growth media and 20 µL/well MTS CellTiter 96® AQueous One Solution. The mixture was incubated for 4 h at 37 °C with 5% CO2. The cell viability was quantified by UV/visible absorbance at 490 nm on a Synergy HT microplate reader (Biotek, Winooski, VT, USA). The Hela cells treated with growth media at corresponding pHs without free DOX or DOX-loaded liposomes were referred to as 100% cell viability.
5
Isolation and Culture of Granulosa Cells
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Follicles were rapidly dissected from each ovary and were then washed with ice-cold phosphate-buffered saline (PBS, Solarbio Science & Technology Co., Ltd, Beijing, China). These follicles were classified into pre-hierarchical (<10 mm) and hierarchical (designated as F5-F1) follicles, and three cohorts of them including the pre-hierarchical (6–10 mm), F4-F2 and F1 follicles were further selected for isolation of GCs based on our previously protocol [35 (link),37 (link)]. GCs isolated from each cohort were digested with 0.1% collagenase (sigma, Aldrich, U.S.A.), counted using the Handheld Automated Cell Counter (Millipore Corporation, Billerica, MA 01821). The GCs were seeded at a density of ∼3 × 105 cells/well (12-well plate) and cultured as previously described [35 (link)], and these cells were used for Oil red O staining and qPCR.
6
Evaluating Antigen-Presenting Cell Recruitment and Activation
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To verify the cell recruitment and activation ability of AM created by microcapsules, microcapsules encapsulating 20 mM NaHCO3 group were evaluated as a control experiment to establish a neutral microenvironment in vivo, and then microcapsules loaded with PBS and NaHCO3 were subcutaneously administrated into animals, respectively, and extracted at day 5. The subcutaneous tissues were cut off extra parts to ensure same weight of tissues. The single-cell suspension was prepared following the method described before. Cells were pelleted and counted using a handheld automated cell counter (Millipore), then FITC-conjugated CD11c and APC-conjugated F4/80 antibodies were managed to analyze DCs and macrophages recruitment, and APC-Cy7-conjugated CD86, eFlour 450–conjugated MHC-I, and Percp-Cy5.5-conjugated MHC-II stains were conducted for DCs and macrophages maturation analysis.
To investigate the influence of antigen dose, adjuvant and particle size on the APC recruitment, activation, and cross-presentation, different vaccine formulations were prepared and injected into C57BL/6J mice. Then, animals were euthanized, and recruited cells were harvested for staining and analyzed by FC at days 7, 14, and 21. Accordingly, subcutaneous nodules were sliced and stained by H&E for histological observation, counted by Inform software, and analyzed.
To investigate the influence of antigen dose, adjuvant and particle size on the APC recruitment, activation, and cross-presentation, different vaccine formulations were prepared and injected into C57BL/6J mice. Then, animals were euthanized, and recruited cells were harvested for staining and analyzed by FC at days 7, 14, and 21. Accordingly, subcutaneous nodules were sliced and stained by H&E for histological observation, counted by Inform software, and analyzed.
7
Immortalized Chicken Preadipocyte Culture and Cloning
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A cell line of immortalized chicken preadipocytes (ICP1)88 (link) was a kind gift of the Poultry Breeding Group of the College of Animal Science and Technology, Northeast Agricultural University, China, and was cultured in DMEM/F12 cell culture medium with 10% FBS, at 37 °C with 5% CO2. ICP1 preadipocytes were seeded in 6-well plates for further transfection using Lipofectamine 2000 (Invitrogen), and the transfection procedure was performed according to the manufacturer’s instructions. After a 48 h recovery period, the cells were supplemented with 3 μg/mL of puromycin (Sigma-Aldrich, MO, USA) to screen out cells which have not been successfully transfected into the plasmids (ie, negative cells). Once the cell clone is formed, cells were harvested using 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA), and the cell density was calculated using a handheld automated cell counter (Millipore, Darmstadt, Germany). Single cells were plated in each well of a 96-well plate by limiting dilution and then cultured for 10 d in the cell culture medium. The medium was replaced every 4 d. Confluent cell colonies were propagated and genotyped by PCR and sequencing. Primer sets used for PCR are listed in Supplementary Table 19 .
8
Isolation of Human PBMCs
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Whole heparinized peripheral blood was diluted 1:2 (vol/vol) in phosphate buffered saline (PBS), pH 7.2. PBMCs were separated on a Ficoll density gradient by centrifugation at 300 g for 30 min at room temperature. After centrifugation, the cells at the interface were collected, washed twice, and counted using a handheld automated cell counter (Millipore Co., Billerica, MA, USA), and viability was assessed by eosin dye exclusion.
9
UVB Damage and LMWP Protection in HaCaT Cells
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HaCaT cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) (containing 10% fetal bovine serum and 1% penicillin/streptomycin) at 37°C in 5% CO2 incubator (bluepard). When the HaCaT cells grew to 80%, they were digested with 0.25% trypsin‐EDTA and counted by handheld automated cell counter (Millipore). Finally, a certified concentration of cell suspension was prepared in the 90‐mm plate for subculture.
The HaCaT cells consisted of three groups: the control group, the UVB model group, and the experimental group, all of which were cultured with 5 × 104 cells/mL in 6‐well plates for 24 h. Control HaCaT cells did not receive any treatment, whereas the oxidative damage model cells received 50 mJ/cm2 UVB irradiation. The cells of the experimental group were first irradiated with 50 mJ/cm2 UVB and then treated with a certain concentration of LMWP for 24 h (LMWP‐1: ten‐thousand‐fold dilution of low molecular weight polypeptides, LMWP‐2: one‐thousand‐fold dilution of low molecular weight polypeptides).
The HaCaT cells consisted of three groups: the control group, the UVB model group, and the experimental group, all of which were cultured with 5 × 104 cells/mL in 6‐well plates for 24 h. Control HaCaT cells did not receive any treatment, whereas the oxidative damage model cells received 50 mJ/cm2 UVB irradiation. The cells of the experimental group were first irradiated with 50 mJ/cm2 UVB and then treated with a certain concentration of LMWP for 24 h (LMWP‐1: ten‐thousand‐fold dilution of low molecular weight polypeptides, LMWP‐2: one‐thousand‐fold dilution of low molecular weight polypeptides).
10
Immortalized Chicken Preadipocyte Differentiation
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A cell line of immortalized chicken preadipocytes (ICPs) [36 (link)] was cultured in DMEM/F12 (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), and 0.2% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). To induce ICPs differentiation, we added 160 μmol/L sodium oleate (Sigma Life Science, St. Louis, MO, USA) to the medium [37 ].
For MYOD1OE and MYOD1KO cell selection, ICPs were seeded in 6-well plates for further transfection using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After a 48-h recovery period, the cells were supplemented with 3 μg/mL of puromycin (Sigma-Aldrich, MO, USA) in the culture medium for 12 days until clone formation. Cells were harvested using 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA), and the cell density was calculated using a handheld automated cell counter (Millipore, Darmstadt, Germany). Single cells were plated in each well of a 96-well plate by limiting dilution and then cultured for 10 d in the cell culture medium. The medium was replaced every 4 d. Confluent cell colonies were propagated and genotyped by PCR and sequencing.
For MYOD1OE and MYOD1KO cell selection, ICPs were seeded in 6-well plates for further transfection using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After a 48-h recovery period, the cells were supplemented with 3 μg/mL of puromycin (Sigma-Aldrich, MO, USA) in the culture medium for 12 days until clone formation. Cells were harvested using 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA), and the cell density was calculated using a handheld automated cell counter (Millipore, Darmstadt, Germany). Single cells were plated in each well of a 96-well plate by limiting dilution and then cultured for 10 d in the cell culture medium. The medium was replaced every 4 d. Confluent cell colonies were propagated and genotyped by PCR and sequencing.
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