Hbss cmf

Manufactured by Merck Group

HBSS-CMF is a balanced salt solution used for the maintenance and washing of cells in cell culture applications. It contains essential inorganic salts, glucose, and phenol red as a pH indicator. HBSS-CMF is calcium and magnesium-free, making it suitable for applications where the presence of these ions is not desired.

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Lab products found in correlation

Poly l lysine coated, by Merck Group (1 mentions) Cmf hbss, by Worthington (1 mentions) Papain, by Worthington (1 mentions) Tc20 automated cell counter, by Bio-Rad (1 mentions) Trypan blue, by Bio-Rad (1 mentions) Dnase 1, by Merck Group (1 mentions) Collagenase, by Roche (1 mentions) Discontinuous percoll gradient, by GE Healthcare (1 mentions) Dispase, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Collagenase 5, by Merck Group (1 mentions) Dnase, by Roche (1 mentions) Cmf hbss, by Thermo Fisher Scientific (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions)

5 protocols using hbss cmf

Dissociation and Fixation of Retinal Cells

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Eyes were prepared as described above. Retinae of one animal were removed from the eyecups, washed in 1 ml warm calcium/magnesium-free HBSS (CMF-HBSS; Sigma Aldrich) for 5 min at 37°C, and incubated for 20 min at 37°C in 1-ml warm papain (Worthington) solution (20 units of papain in 1-ml CMF-HBSS). papain action was stopped by washing the retinae twice in warm HBSS. The tissue was gently dissociated in 1 ml fresh HBSS by pipetting 15–20 times using a cut and fire-polished 1000-μl plastic tip (Nerbe plus). After dissociation, cells were seeded and settled for 30 min on poly-L-lysine-coated (Sigma) coverslips at RT in a humidified chamber before fixation (5 min at RT with 4% paraformaldehyde). The fixative was then removed and cells were washed in PB (twice for 10 min each).

Gehlen J., Aretzweiler C., Mataruga A., Fahlke C, & Müller F. (2021). Excitatory Amino Acid Transporter EAAT5 Improves Temporal Resolution in the Retina. eNeuro, 8(6), ENEURO.0406-21.2021.

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Isolation and Characterization of Polymorphonuclear Leukocytes

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Isolation of polymorphonuclear leukocytes, viability, and cell differentiation were performed using endotoxin free materials and reagents, according to the procedures described by Garcia et al. (2015) . Briefly, after blood centrifugation, plasma and one-third of the red blood cells (RBC) were discarded and the remaining RBCs were lysed with ice-cold water and isotonicity was restored using PBS. The pellet was washed 3 times in 10 mL of PBS and resuspended in 1 mL of calciumand magnesium-free Hanks' buffered saline solution (CMF-HBSS; Sigma-Aldrich Co., St. Louis, MO). Concentrations of PMNL were measured using a TC-20 automated cell counter (Bio-Rad Laboratories Inc., Hercules, CA). The viability of PMNL was determined using the trypan blue (0.1%, Bio-Rad Laboratories Inc.) exclusion method, resulting in an average viability of 94%. Cell differentials were determined microscopically on cytospin preparations using a commercially available hematology staining kit (Hema-FastTM 3-Step Hematology Staining Kit; Fisherbrand, Thermo Fisher Scientific Inc.), indicating that 85% of the cells were PMNL.

Garcia M., Elsasser T.H., Qu Y., Zhu X, & Moyes K.M. (2015). Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro. Journal of dairy science, 98(9).

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Isolation of Intestinal Immune Cells

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MLN, Peyer’s patches, intraepithelial lymphocytes (iEL) and lamina propria lymphocytes (LPL) were isolated by the method of Lefrancois and Lycke ³⁷. Briefly, MLN were isolated and then the intestine from the duodenum to the rectum was flushed with Ca²⁺Mg²⁺-free 100 mM HBSS (CMF) (Sigma-Aldrich, St. Louis, MO). The colons were cleaned by removal of fat, fecal matter and debris. RPMI was injected into the intestine to highlight the Peyer’s patches, which were then collected with a fine forceps and scissors. The intestines were opened longitudinally and cut into 0.5 cm sections, shook twice with CMF solution containing 10 mM HEPES, 25 mM NaHCO₃, 1 mM DTT and 1 mM EDTA all from Sigma-Aldrich, St. Louis, MO, and 2% fetal bovine serum (FBS) Gibco®, Portland, OR at 37 °C. IELs were then purified using discontinuous Percoll gradients (GE Healthcare, Buckinghamshire, UK) by collecting mononuclear cells at the interface between the 40% and 70% layers. For the isolation of LPLs, intestinal pieces were digested for 70 min with complete RPMI-1640 containing 1 mg/ml collagenase (Roche Applied Science, Upper Bavaria, Germany), 40 μl/ml dispase (Sigma-Aldrich, St. Louis, MO) and 4 μl/ml Dnase I (Sigma-Aldrich, St. Louis, MO) at 37 °C. LPLs were then purified using 40%/70% discontinuous Percoll gradients.

Wang L., Ray A., Jiang X., Wang J.Y., Basu S., Liu X., Qian T., He R., Dittel B.N, & Chu Y. (2015). T regulatory cells and B cells cooperate to form a regulatory loop that maintains gut homeostasis and suppresses dextran sulfate sodium-induced colitis. Mucosal Immunology, 8(6), 1297-1312.

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Isolation of Lamina Propria Mononuclear Cells

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LPMCs were isolated from freshly obtained biopsies using a previously described DDT-EDTA collagenase method (13 (link), 14 (link)). Briefly, biopsies were washed in HBBS free of calcium and magnesium (HBSS-CMF; Hyclone, Europe LTD, Cramlington, United Kingdom), and then incubated for 5 min at room temperature in HBSS-CMF containing 1 mmol/l DTT (Sigma Chemical Co., St. Louis, MO, United States) and antibiotics (penicillin, 100 U/ml; streptomycin, 100 mg/ml; gentamicin, 50 mg/ml; and fungizone, 25 mg/ml). After washing 3 times in HBSS-CMF, the biopsies were cut into smaller pieces and incubated in HBSS-CMF containing 0.75 mmol/l EDTA, 10 mmol/l HEPES buffer, and antibiotics for 15 min at 37°C in a humid 5% CO₂ atmosphere to remove epithelial cells. After 2 washes, the tissue was incubated for a total of 2 h (2 × 1-h incubations) at 37°C in a humid 5% CO₂ atmosphere in complete medium (RPMI 1640 plus 10 mM HEPES buffer, 2 mM l-glutamine, 10% heat-inactivated FCS (Hyclone), and antibiotics) containing 25 U/ml collagenase V (Sigma-Aldrich, Milan, Italy) and 100 μg/ml of DNase (Roche Diagnostics, Mannheim, Germany). After incubation, the supernatant containing LMPCs was collected and washed twice in HBSS-CMF + antibiotics.

Butera A., Sanchez M., Pronio A., Amendola A., De Nitto D., Di Carlo N., Lande R., Frasca L., Borrini F., Pica R, & Boirivant M. (2018). CD3+CD4+LAP+Foxp3-Regulatory Cells of the Colonic Lamina Propria Limit Disease Extension in Ulcerative Colitis. Frontiers in Immunology, 9, 2511.

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Aggrecan Gradients for Neurite Outgrowth

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Delta-T cell culture dishes (Fisher) were prepared as previously described58 (link). Culture dishes were rinsed with sterile water and then coated with poly-L-lysine (0.1 mg/ml, Sigma-Aldrich) overnight at room temperature, rinsed with sterile water, and allowed to dry. Aggrecan spot gradients were formed by allowing 2 μl of aggrecan solution (0.7 mg/ml in HBSS-CMF, Sigma-Aldrich) to dry onto the culture surface. The surface of the dish was bathed in laminin solution (10 μg/ml in HBSS-CMF, Invitrogen) for 3 hr at 37°C. The laminin bath was subsequently removed and cells were plated without allowing the surface of the dish to dry.

DePaul M.A., Palmer M., Lang B.T., Cutrone R., Tran A.P., Madalena K.M., Bogaerts A., Hamilton J.A., Deans R.J., Mays R.W., Busch S.A, & Silver J. (2015). Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury. Scientific Reports, 5, 16795.

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