9L rat glioma cells (ATCC) were grown in Dulbecco's Modified Eagle Medium (DMEM; Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah) and antibiotics (100 units/ml penicillin and streptomycin, Mediatech, Inc., Herndon, VA). Glioma cells (3×105 9L) were transfected with the luc gene using the PGL3-luc vector (2 µg; Promega, www.promega.com ) using Fugene 6 (Roche Diagnostics, Indianapolis, IN) and selected for highest expression of the luc gene using 400 µg/ml G-418 disulfate (RPI, Research Products Inc., Mt. Prospect, IL). A high expression stable single cell clone was selected and was transfected with a retrovirus to constitutively express GFP (Invitrogen, www.invitrogen.com ) and selected using 10 µg/ml puromycin dihydrochloride (Sigma, St. Louis, MO). Further transfection was induced with the mCherry virus (Clontech, www.clontech.com ). High expressing clones of 9L-luc-GFP and 9L-luc-GFP-mCherry cells were isolated using Flow Cytometry (BD Facscallibur, San Jose, CA). The resultant cells were cultured in DMEM and supplemented with 200 µg/ml G418 and 3 µg/ml puromycin. Cell counts were performed using a hemocytometer (Bio- Rad, Philadelphia, PA) and with Trypan blue exclusion. Tumor volumes were measured using electronic calipers (Global Industrial, Port Washing ton, NY)
Hemocytometer
Manufactured by Bio-Rad
Sourced in United States
A hemocytometer is a device used for counting cells, such as blood cells, in a sample. It consists of a thick glass slide with a chamber of known depth and area, allowing the volume of the sample to be precisely measured. This enables the calculation of the number of cells per unit volume.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pgl3 luc vector, by Promega (1 mentions)
Flow cytometry, by BD (1 mentions)
Puromycin dihydrochloride, by Merck Group (1 mentions)
Trypan blue dye, by Bio-Rad (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
System 2 software, by BD (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Diff quick, by Solarbio (1 mentions)
F 12k medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Tc20 counter, by Bio-Rad (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypan blue staining, by Thermo Fisher Scientific (1 mentions)
Ad293 cells, by Cell Biolabs (1 mentions)
8 protocols using hemocytometer
1
Generating Stable Glioma Cell Lines for Tumor Studies
2
Flow Cytometry Analysis of Splenic T Cells
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Splenic single-cell suspensions prepared from all mice prior to the challenge were used to analyze T lymphocytes using flow cytometry, as previously described [22 (link)]. Briefly, 105 viable cells counted using a hemocytometer and 0.04% trypan blue dye (Bio-Rad) were suspended in 100 μL of 2% FBS in PBS solution and incubated with 0.45 μL of FITC anti-mouse CD3e, 0.25 μL of APC-eFluor780 anti-mouse CD4 or 0.25 μL of Percp-cy5.5 anti-mouse CD8a (eBioscience, San Diego, California, USA) at 4 °C for 30 min in the dark. After two washes with 2 mL of PBS, the samples were re-suspended in 300 μL of FACScan buffer (0.1% NaN3, 1% BSA) and 250 μL of 2% PFA solution for optical measurement. The data from three independent experiments were obtained using the FACScan flow cytometer and SYSTEM II software (BD Bio-sciences, San Jose, California, USA).
3
Bronchoalveolar Lavage Fluid Analysis
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Samples of BALF were collected 24 h following the final challenge, according to previously described methods. The lungs were lavaged with 1.6 ml (0.8 ml per lavage) of ice-cold PBS via the tracheal cannula. The retrieved volume (~75–80%) of the instilled PBS was then centrifuged at 500 × g at 4°C for 5 min. The resultant pellet was washed twice with PBS and was resuspended in 1 ml PBS. The total numbers of cells were counted using a hemocytometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Smears of the BALF cells were stained with Wright and Giemsa staining fluid (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for differential cell counting. Using a BX-60 Olympus microscope, the numbers of cells were counted by two independent investigators, in a blinded-manner. A total of ~200 cells were counted in each of four randomly selected locations.
4
Culturing and Quantifying GM12878 Cells
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GM12878 lymphoblastoid cells were obtained from Coriell Cell Repositories and maintained in RPMI 1640 with 1 % Penstrip and 10 % FBS. Cells were kept in suspension at 37 °C and were harvested when suspensions reached optimal density. Cell numbers were quantitated with a hemocytometer (Biorad), pelleted at 250 × g for 4 min and washed twice with a volume of PBS sufficient to obtain approximately 1,000,000 cells per mL in approximately 1 mL of PBS, and diluted as needed in PBS for experiments. All biological replicates were performed with material from the same cell culture flask, with independent running of the assays. When cells were diluted to 100,000 cells per mL they were quantitated again on the hemocytometer.
5
Rat Lung Lavage and Blood Collection
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The rats were anesthetized and humanely killed 24 h after the last challenge. Blood samples were collected from the abdominal aorta and the supernatants were centrifuged at 1,006 g for 15 min and stored at –80 °C. The rats’ left lungs were washed with saline solution via a tracheal cannula, and the fluid collected was centrifuged at 112 g for 15 min at 4 °C. The supernatants were kept as BALF and stored at –80 °C. The cell pellet was resuspended in saline solution using a hemocytometer (Bio-Rad, USA) to count the total number of cells. Wright’s staining was performed to obtain differential cell counts.
6
Cell Count and Differential Analysis
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After resuspending the cell pellet using 200 μL PBS, a hemocytometer (BioRad, Shanghai, China) was applied to count total cells in 50 μL cell suspension. Another 50 μL cell suspension was stained by Diff‐Quick (Solarbio, Beijing, China) to examine differential cell counts. The percentages of eosinophils, neutrophils, and lymphocytes in BALF were determined by counting 500 leukocytes in randomly selected fields under a light microscope.
7
Biocompatibility Evaluation of HUCs on PP and PP-gel Scaffolds
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HUCs were cultured in F12K medium (Gibco, United States) containing 10% fetal bovine serum (FBS, Gibco), 100U/mL penicillin, and 100 g/ml streptomycin (Gibco) at 37 °C in a humidified environment with 5% CO2. The medium was renewed every two days and subcultured when cells were confluent around 80%. For the biocompatibility tests, HUCs were seeded on PP and PP-gel scaffolds. A cell counting kit-8 assay (CCK-8) was used to detect the cell metabolic activity (n = 5), which was directly correlated with the cell number. At the same time, the cell number was counted using a hemocytometer (Bio-Rad, United States). A confocal laser microscope (CLSM, SP8, Leica, Germany) and a scanning electron microscope (SEM) were used to observe the distribution of HUCs on scaffolds. The cells were stained with F-actin and the nuclei were stained with phalloidin/4, 6-diamidino-2-phenylindole (DAPI).
8
Cultivation of Mouse Insulin-Producing Cells
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Mouse MIN647 (link) and βTC48 (link) β-cells were maintained in DMEM containing 10% fetal bovine serum (FBS) (all from ThermoFisher Scientific, Waltham, MA) at 37 °C and 5% CO2. Cells were split 1:4 at 70–90% confluence. PIs were formed by seeding 2–5 × 104 cells/ml onto Petri dishes or 105 cells/ml in 125-ml ProCulture spinner flasks29 (link) (Corning, NY). In spinner flask PI cultures, half-volume medium changes were performed every 2–3 days. AD293 cells (Cell Biolabs Inc., San Diego, CA) used for recombinant adenovirus production were cultured in the same medium as above but supplemented with 1% MEM non-essential amino acids (ThermoFisher). Cell viability was determined by Trypan Blue staining (ThermoFisher) using a hemocytometer or a TC20 counter (Bio-Rad Laboratories, Hercules, CA).
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