Endometrial stromal cells were isolated from ectopic, eutopic, and normal control endometrial tissues and were named ESCs, EuSCs, and NESCs, respectively, as described in a previous study [21 (link)]. Samples were washed twice with phosphate-buffered saline (PBS), minced, and coated with 1 mg/ml collagenase type IV (BS165, Biosharp, The Netherlands). Samples were incubated at 37 °C with mild shaking for two hours in a culture flask. Next, a 40-mm sieve was used to remove debris and other cells, such as endometrial epithelial cells, and the solution that passed through was centrifuged for 10 min at 1000 r/min. Cell precipitates were collected by removing the supernatant. The cells were grown in a 5% CO2 humidified atmosphere at 37 °C in DMEM/F12 (MA0214, Meilunbio, China) supplemented with 10% foetal bovine serum (BI, Israel).
Bs165
Manufactured by Biosharp
Sourced in China
The BS165 is a centrifuge designed for general laboratory use. It features a compact design and a fixed-angle rotor with a maximum speed of 6,000 RPM. The centrifuge can accommodate a variety of sample sizes and volumes, making it a versatile piece of equipment for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Foetal bovine serum (fbs), by Meilun (1 mentions)
Dmem f12, by Meilun (1 mentions)
Bs 70 cs, by Biosharp (1 mentions)
Bs909, by Biosharp (1 mentions)
Hank s balanced salt solution, by Merck Group (1 mentions)
Red blood lysis buffer, by Biosharp (1 mentions)
Hanks balanced salt solution (hbss), by Biosharp (1 mentions)
Bs163, by Biosharp (1 mentions)
40 μm strainer, by Corning (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
3 protocols using bs165
1
Isolation of Endometrial Stromal Cells
2
Isolation of Liver Lymphocytes
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Mice were anesthetized by inhaling isoflurane, the portal of the mice was exposed, and a soft catheter was inserted into the portal vein of the mice. The prewarmed Hank’s balanced salt solution (Sigma-Aldrich, MO, USA) was pumped into the liver to remove residual blood. And the livers were cut up and digested in type IV collagenase (BS165, Biosharp, Hefei, China) for 1 h and ground in a 70 μm cell strainer (BS-70-CS, Biosharp, Hefei, China) to obtain a cell suspension, and the cells were subsequently centrifuged at 700g for 5 min at 4 °C. After centrifugation, cell suspension was re-suspended with 40% percoll (BS909, Biosharp, Hefei, China) solution and slowly added to 70% percoll solution for gradient centrifugation to separate lymphocytes. The collected lymphocytes are washed and centrifuged for subsequent detection.
3
Isolation and Processing of Tumor and Normal Lung Cells for scRNA-seq and IHC
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Immediately following collection, tumor tissues and distal normal lung tissues were immersed in Hank’s balanced salt solution (HBSS, Gibco), and rapidly transported to laboratory at a low temperature. These samples were quickly divided into two pieces; one-half was treated with enzymatic digestion and cell sorting for scRNA-seq, and the other was fixed in 10% neutral formalin solution and paraffin-embedded for TRS, IHC and mIHC staining.
For preparing single cells, tissues were minced with scissors on ice and digested well with 1 mg/mL collagenase I (BS163, Biosharp), 0.5 mg/mL collagenase IV (BS165, Biosharp) and HBSS. After 30 min of incubation at 37°C on a shaker, samples were filtered using the 40μm strainers (Corning), centrifuged at 500 x g for 5 min at 4°C and then removed the supernatant. The remaining cell pellet was treated with red blood lysis buffer (Biosharp) for 5 min to remove the red blood cells, followed by centrifugation once more for 5 min (500g, 4°C). After the removal of the supernatant, the samples were resuspended in the sorting buffer (0.04% BSA and PBS). The dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec, Germany) according to the manufacture’s protocol. The single-cell suspensions with a rate of cell viability greater than 80% were handled according to manufacturer’s instructions subsequently for scRNA-seq.
For preparing single cells, tissues were minced with scissors on ice and digested well with 1 mg/mL collagenase I (BS163, Biosharp), 0.5 mg/mL collagenase IV (BS165, Biosharp) and HBSS. After 30 min of incubation at 37°C on a shaker, samples were filtered using the 40μm strainers (Corning), centrifuged at 500 x g for 5 min at 4°C and then removed the supernatant. The remaining cell pellet was treated with red blood lysis buffer (Biosharp) for 5 min to remove the red blood cells, followed by centrifugation once more for 5 min (500g, 4°C). After the removal of the supernatant, the samples were resuspended in the sorting buffer (0.04% BSA and PBS). The dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec, Germany) according to the manufacture’s protocol. The single-cell suspensions with a rate of cell viability greater than 80% were handled according to manufacturer’s instructions subsequently for scRNA-seq.
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