Isolation of Nucleated Cells from Whole Blood v1

This protocol describes a method for the isolation of pan-lymphocytes and pan-myeloid cells from human whole blood. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Isolation of Nucleated Cells from Bone Marrow Aspirate v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human bone marrow aspirate. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

​Sex Ratio of Calves Resulted from Artificial Insemination Implementation using Sexed Semen with Percoll Gradient Density Centrifugation Method in Ongole Crossbred Cows

Background: Productivity of existing cattles in Indonesia is necessarily to be increase to balance meat consumption in this country. Determination of offspring of certain sex can be obtained from Percoll gradient density centrifugation.The purpose of this research was to elucidate the proportion of male calves that can result from artificial insemination using single and double doses of sexed semen in Ongole crossbred cows. Methods: The sexed semen samples were obtained through Percoll gradient density centrifugation performed by the Artificial Insemination Center. The artificial insemination method adopted here was deep insemination. As much as 10 ml of BIO ATP® (Rheinvet) was injected in each cow before immediately insemination. Further, as much as 3 kg/day of additional feed was given over three days after insemination, with a protein level of about 12%. Result: Our results showed the proportion of Y-bearing sperm among non-sexed semen was 52.77% and among sexed semen, was 80.79%. Further, 54.17% of the non-sexed semen, 42.11% of the single-dose sexed semen and 78.95% of the double-dose sexed semen treatments yielded male calves.

Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60–95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiationin vitrowere preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

Pemisahan Spermatozoa Berkromosom X dan Y Kambing Boer dan Aplikasinya Melalui Inseminasi Buatan Untuk Mendapatkan Jenis Kelamin Anak Sesuai Harapan

Separation of spermatozoa with x and y chromosome at boer goat and its application by artificial insemination for kid sex purposeABSTRACT. The purposes of this experiment are to investigate the separation of X and Y spermatozoa by measuring the spermatozoa quality, sex ratio between X and Y, capacity of fertility indicated by conception rate and sex ratio of goat boar kids. Samples, used in this experiment, are fresh semen from Boer goat with high quality consists of 4 group treatments with 6 replications 1) group of spermatozoa without separation (control), 2) group of spermatozoa separated by percoll gradient density centrifugation 3 levels (P1), 5 levels (P2) and swine up (P3). The observed parameters are spermatozoa quality, X and Y spermatozoa ratio, fertility’s capacity and sex ratio on the birth. Quality examination of spermatozoa and identify X and Y spermatozoa is based on the standard method of WHO. The conception rate was based on the ratio of pregnant goat after the first insemination. Data of spermatozoa quality and spermatozoa ratio were analyzed by using analisis of variance (ANOVA) and further analysis by LSD if there were differences between treatments. The results of this experiment showed that spermatozoa quality Boer goat significantly reduced (p0,05) after separation with percoll gradient density centrifugation and swim up. Percentage spermatozoa X after percoll gradient density centrifugation was significantly higher (P0,05) compared to control and swim up. Meanwhile, the Y spermatozoa population was significantly higher (P0,05) after swim up treatment compared to percoll gradient density centrifugation and control. The percentage of sex ratio (male: female) after insemination from percoll gradient density centrifugation produced more female than male. On the other hand, insemination from swim up produced more male than female. Sex ratio produced from separation of percoll gradient density centrifugation, swim up was difference from control semen (P0,05). From this experiment, it was concluded that spermatozoa separation by percoll gradient density centrifugation and swim up can be used as one of the methods to separate X and Y spermatozoa and further can be applied to get preferred sex animals.