Chicken splenocytes were isolated from spleens collected from healthy birds, under laminar air flow as per standard procedure [13 (link)]. Lymphocytes were separated by density gradient centrifugation (Hisep-LSM, HiMedia, India) as per the method described by Rose and Friedman [14 ]. Percentage cell viability was determined by 0.1% Trypan blue dye exclusion test using hemocytometer [15 (link)], and final cell count was adjusted to 107 cells/ml in RPMI-1640 medium with antibiotic and antimycotic solution supplemented with 10% Fetal Bovine Serum (all HiMedia, India).
Hi sep lsm
Manufactured by HiMedia
Sourced in India
The Hi-Sep LSM is a laboratory centrifugation product designed for the separation and isolation of cells, particles, and other biological components. It functions as a tool for sample preparation and processing in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by HiMedia (1 mentions)
Rpmi 1640 medium, by HiMedia (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Sodium heparin vacutainer, by BD (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
L glutamine, by HiMedia (1 mentions)
Rpmi 1640, by PAN Biotech (1 mentions)
6 protocols using hi sep lsm
1
Isolation and Characterization of Chicken Splenocytes
2
Generating Myeloid Adherent Cells from hPBMC
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Human blood was drawn from healthy donors following the guidelines of the Institutional Ethics Committee, NISER, Bhubaneswar (NISER/IEC/2022-04). The procedure for generating myeloid adherent cells from human peripheral blood mononuclear cells (hPBMC) was followed as described elsewhere with little modifications (38 (link)–41 (link)). Briefly, circulating monocytes were enriched by 2 h adherence after Hi-Sep LSM (catalog number: HiSep LSM™ 1077‐ LS001; HiMedia Laboratories Pvt Ltd, India) based density gradient-centrifugation according to the manufacturer’s instructions. The adherent cells were cultured in RPMI-1640 supplemented with 10% FBS, antibiotic-antimycotic solution and L-glutamine for 3–5 days. The adherent cells obtained after 96 h were of monocyte-macrophage lineages (more than 97%) as found enriched with CD14+CD11b+ population (42 , 43 (link)). The monocyte-macrophage cells derived from hPBMC were seeded in 12 well plates (Thermo Fischer, USA) at a density of 0.8x106 cells/well. After 24 h of seeding, pre-incubation was carried out for 3 h with 1 µM of TAK-242, followed by CHIKV infection with MOI 5 for 2 h (19 (link)). The infected cells were harvested at 8 hours post-infection (hpi) and downstream experiments were conducted.
3
Comet Assay for DNA Damage
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The method of Singh et al.[33 (link)] was followed with minor modification. The lymphocyte cells were isolated from blood of four groups of mice using reagents and protocol provided by HiSep LSM (Hi Media, Germany). The cells were suspended in 110 μl of low melting point agarose (0.65% LMPA – W/V in phosphate-buffered saline [PBS], pH = 7.4) and smeared on a frosted glass microscope slide precoated with 140 ml of 1% normal melting point agarose (in PBS, pH 7.4). The agarose was allowed to set for 10 min at 4°C and thereafter the coverslip was removed. The slides were exposed to lysis solution overnight (2.5M NaCl; 0.1M Na2 HPO4; 10 mM Tris-Cl; 0.3M NaOH; 1% Triton ×100; 10% dimethyl sulfoxide pH 10). The solution was kept at 4 C for 1 h before use. On next day, slides were transferred into a horizontal electrophoresis chamber containing electrophoresis buffer (300 mM NaOH, 1 mM Na2-EDTA; pH 13.0) and presoaked for 20 min to unwind DNA. Electrophoresis was carried out for 20 min (30 mA, 20 V). Slides were then washed thoroughly with neutralizing buffer (Tris 0.4M, pH 7.5), stained with Et Br (final concentration of 40 μg/ml), and examined under a Leica fluorescence microscope. The DNA breakage percentage was determined by measuring the COMET tail length using the software Motic Image China.
4
Isolation and Culture of Mesenchymal Cells
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Mice were sacrificed by CO2 asphyxiation and femurs extracted aseptically. These bones were flushed using plain DMEM; the cellular suspension was loaded onto HiSep LSM (HiMedia, Mumbai, India) and centrifuged for 30 min at 1500 rpm and room temperature. Buffy coat was aspirated and washed two times with plain DMEM for 15 min at 1500 rpm and room temperature. Then, these cells were enumerated, and complete mononuclear cell (MNC) fraction (viability >98% as determined by trypan blue dye exclusion test) was seeded onto PCG matrix and cultured in a medium comprising 1:1 DMEM + 10% mesenFBS + EGM-2 + 20% FBS at 37°C, 5% CO2 for 14 days. The medium was replenished every 48 h. After 14 days of culture, CM was collected separately and the entrapped cell secretome harvested mechanically using sterile glass rolling mechanical extractor by gently pressing the matrix. The collected secretome and CM were stored at -80°C.
This PCG formulation and culture process for MCS formation are under IPR (IPA no.1951/MUM/2014).
This PCG formulation and culture process for MCS formation are under IPR (IPA no.1951/MUM/2014).
5
Comparing Blood and TM Tissue in Glaucoma
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Since the TM tissue is bathed with aqueous humor, comparing changes in the peripheral blood versus the tissue changes would give an insight if the local tissue changes may represent an effect of peripheral stimuli or a local response restricted to the TM tissue only. We therefore compared the gene expression in the peripheral blood of these patients whose ex-vivo dissected TM specimens were analyzed. Whole blood was collected from patients whose TM specimens were collected (POAG, PACG and control (cataract)) into EDTA tubes. In addition, those with moderate stable glaucoma (not undergoing surgery) were also analyzed for gene expression in peripheral blood to compare with those with severe glaucoma. Whole blood was diluted with 1X phosphate- buffered saline (pH 7.2) in the ratio of 1:1. Aseptically 2.5 mL of Hisep LSM (Himedia) was overlaid with 7.5 mL of diluted blood in the proportion of 1:3 followed by centrifugation at 400 x g at room temperature for 30 minutes to separate the lymphocyte layer which was carefully aspirated. Equal volume of 1X PBS was added and mixed by gently aspiration and centrifuged at room temperature at 300 x g for 10 minutes. Cell death analysis by FACS and gene expression for cell death-related genes (described above) was carried out in PBMC. Immunoblotting was used to study histone modifications in PBMC as detailed earlier.
6
Isolation and Culture of hPBMCs
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Isolation of human peripheral blood mononuclear cells (hPBMCs) was performed according to the procedure described earlier. Briefly, 30 mL blood was collected from each of three healthy individuals in sodium heparin Vacutainers (BD, NJ). Hi-Sep LSM (HiMedia, India) was used to isolate PBMCs from the blood samples. Isolated PBMCs were cultured in RPMI 1640 (PAN Biotech) supplemented with 15% FBS (PAN Biotech), l -glutamine (HiMedia, India), and antibiotic-antimycotic solution for 5 days. Every 2 days, the cells were washed and supplemented with fresh medium (58 (link)).
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