Sample pretreatment for flow cytometry analysis was conducted according to a previous study (Fang et al. 2006 (link)). Five milliliters of cells harvested from ePBR culture were pelleted and resuspended in 5 ml of ethanol/acetic acid (3:1) solution for fixation for 2 h at room temperature. After fixation, cells were rinsed twice and resuspended with 1 ml of FACS buffer containing 0.2M Tris–HCl (pH 7.5) and 20 mM EDTA and stored at 4°C. Prior to flow cytometry analysis, the cell density of each sample was measured with a Z2 COULTER COUNTER Analyzer (Cat. 6605700, Beckman Coulter), and all samples were normalized to 1 × 106 cells/ml in a total 900 µl of FACS buffer, mixed with 100 µl of FACS buffer containing 1 mg/ml of RNase A, and incubated at 37°C for 2 h. For DNA staining, the RNase A-treated samples were rinsed once and resuspended in 1 ml of PBS buffer. Four hundred and ninety-five microliters of sample were mixed with 5 µl of 100-fold diluted SYTOX Green (cat. S7020, Thermo Fisher Scientific) for 5 min. Flow cytometry was performed with the LSR II Flow Cytometer System (BD Biosciences) at the MSU Flow Cytometry Core (https://facs.iq.msu.edu/ ; accessed 2022 February 7), and data were analyzed with FACSDiva Software (BD Biosciences).
Z2 coulter counter analyzer
Manufactured by Beckman Coulter
Sourced in United States
The Z2 Coulter Counter Analyzer is a versatile instrument used for particle counting and sizing. It employs the Coulter principle to precisely measure the volume and number of particles suspended in an electrolyte solution. The analyzer can be used to count and size a wide range of particles, including cells, bacteria, and other microscopic entities.
Automatically generated - may contain errors
Lab products found in correlation
Cyquant cell proliferation assay, by Thermo Fisher Scientific (2 mentions)
3d cell titerglo assay, by Promega (2 mentions)
Glomax plate reader, by Promega (2 mentions)
Gentamicin, by Sandoz (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Echo 550, by Beckman Coulter (2 mentions)
Celltiter glo, by Promega (2 mentions)
Lsr 2 flow cytometer system, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cls3421, by Merck Group (1 mentions)
Mab678 sp, by R&D Systems (1 mentions)
Mab672 sp, by R&D Systems (1 mentions)
Isoton 2 diluent, by Beckman Coulter (1 mentions)
Trypsin edta, by Corning (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Evos xl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
Isoton 2, by Beckman Coulter (1 mentions)
25 protocols using z2 coulter counter analyzer
1
Flow Cytometry Sample Preparation
2
Chemokine-mediated Jurkat cell migration
3
Myeloma Cell Culture Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OPM-1, RPMI-8226, NCI-H929, MM1S, and U266 cells were cultured in six-well plates. The initial cell number was 100,000 cells per well and the cell numbers were counted and re-plated at the initial concentration every 3 days. Lenalidomide and AI-10-104 were added into the culture media after dilution every 3 days. The cell counting was conducted with Z2 Coulter Counter Analyzer (Beckman Coulter, #6605700).
4
Cell Volume Measurement Technique
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One confluent T75 flask was used for each volume measurement experiment. When needed, drug preincubations were performed on detached cells inside the cell incubator for the indicated time. All volume measurements were performed at RT, keeping the cells in warm complete medium. A Z2 Coulter Counter Analyzer (Beckmann) was used to measure the volumes of the cell populations. Each sample taken by the counter contained between 20,000 and 100,000 cells in a volume of 100 or 500 μL. Three baseline measures were taken in normal (isotonic) medium before adding the hypertonic or hypotonic medium. Changes in cell volume were normalized to basal volumes. To calculate the percentage of RVD or RVI, the Δ of mean basal volume was calculated to the volume at each time point. Maximum (swollen cells) or minimum (shrunken cells) cell volume changes were set as 100% and subsequent values were referred to this maximum/minimun to calculate the percentage of recovery (%RVI) (62 (link)).
5
Cytotoxicity Evaluation of Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB 231 breast cancer cell line was seeded at a density of 1.75 × 105 cells in a 24-well plate. Twenty-four hours after seeding, cells were transduced as described above. Cytotoxicity was assessed by growth inhibition assay after a 24 h exposure to cisplatin (100 μM), doxorubicin (10 μM) or taxol (50 μM). Each experimental sample was run in quadruplicate. At the end of treatment, each well was washed with PBS to remove dead cells and debris. The remaining cells were detached from plate using Trypsin/EDTA (Corning, New York, NY, USA), resuspended in ISOTON® II Diluent (Beckman Coulter Life Sciences, Indianapolis, Indiana, IN, USA) and counted using Z2™ COULTER COUNTER® Analyzer (Beckman Coulter Life Sciences).
6
Cell Viability Assays for 2D and 3D Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability of 2D-cultured cells was determined using a Z2 Coulter Counter Analyzer (Beckman) or by the CyQuant cell proliferation assay (Invitrogen). Cell viability of 3D-cultured organoids was determined by the 3D Cell TiterGlo assay (Promega) on a GloMax plate reader (Promega).
7
Matrigel Invasion Assay for Cell Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected in 60 mm tissue culture plates for 24 hours. Harvested cells were counted on the Z2 Coulter Counter Analyzer (Beckman Coulter). Cells were then resuspended in Dulbecco's Modified Eagle Medium (Sigma-Aldrich) without serum. The resuspended cells were then pipetted into a Corning BioCoat Matrigel Invasion Chamber (ThermoFisher), 8 μm pore size, in 24-well tissue culture-grade plates. Dulbecco's Modified Eagle Medium (Sigma-Aldrich) supplemented with 10% FBS (Atlas Biologicals) was placed in the well of the plate as a chemo-attractant. After 24 hours, non-invading cells were removed using a cotton-tipped swab. Invasive cells, embedded into the Matrigel, was fixed using 70% ethanol then stained with a 0.5% crystal violet solution. Microscopy was performed with the Evos XL Cell Imaging System (ThermoFisher). Cells were counted in five fields of view at 10× magnification and then averaged as cells per 10× field.
8
Cell Growth and Drug Sensitivity Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For growth assays, 2 × 104 − 3 × 104 cells were plated in each well of a 6-well plate in triplicate. Wells were counted on subsequent days using a Z2 Coulter Counter Analyzer (Beckman Coulter). Population doublings were calculated using the following formula: 3.32 x [log(the number of cell harvested)–log(the initial number of cells plated)].
For sensitivity assays, 2.5 × 104 – 4.5 × 104 cells were plated in each well of a 6-well plate in triplicate. The following day drugs were added at indicated concentrations. After 5-6 days in culture, cells were passaged once at appropriate ratios. Cells were counted when untreated wells reached near confluence around 7-9 days. The cell numbers at each dose of drug were divided by the cell number in the untreated sample to calculate the percent survival.
For sensitivity assays, 2.5 × 104 – 4.5 × 104 cells were plated in each well of a 6-well plate in triplicate. The following day drugs were added at indicated concentrations. After 5-6 days in culture, cells were passaged once at appropriate ratios. Cells were counted when untreated wells reached near confluence around 7-9 days. The cell numbers at each dose of drug were divided by the cell number in the untreated sample to calculate the percent survival.
9
Dendritic Cell-T Cell Co-culture Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDCs were cultivated from fresh or immortalized (Drobek et al., 2018 (link), Ruedl et al., 2008 (link)) hematopoietic stem cells. The dendritic cells were pulsed with indicated concentration of indicated peptides and mixed with isolated T cells or thymocytes in ratio 1:1 or 1:2 and co-cultured in RPMI (Sigma Aldrich) containing 10% FBS (GIBCO), 100 U/ml penicillin (BB Pharma), 100 μg/ml streptomycin (Sigma Aldrich), 40 μg/ml gentamicin (Sandoz) for indicated period of time (pERK1/2 analysis) or over-night (CD25 and CD69 analysis). The EC50 values for the CD69 upregulation assay were calculated using non-liner fit (log(agonist) versus response–Variable slope (four parameters)) in GraphPad Prism 5. The cell numbers were counted using Z2 Coulter Counter Analyzer (Beckman Coulter).
10
Measuring Bacterial Growth Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optical density of aerobic cultures was measured at 660 nm (OD660) with a Jenway 7200 Spectrophotometer (Bibby Scientific, Staffordshire, UK). Maximum specific growth rates were calculated from the slope of ln OD660 versus time during the exponential phase, considering at least six time points. Optical density measurements in anaerobic cultures were performed at 600 nm with an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience, Holliston MA) placed in the anaerobic workstation. For spot-plate experiments, cell counts in late-exponential-phase shake-flask cultures were first determined using a Z2 Coulter Counter Analyzer (Beckman Coulter Life Sciences, Indianapolis IN) following the manufacturer’s protocol. Prior to analysis, cultures were diluted one 100-fold with Isoton II (Beckman Coulter, Brea CA), followed by five replicate cell counts on each sample.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!