Human PBMCs were isolated from healthy donor (Blood Bank, University Hospital Basel, Switzerland) whole blood by density gradient centrifugation, using Histopaque (Sigma-Aldrich, 10771), and then frozen in liquid nitrogen, using fetal bovine serum (FBS) (Pan Biotech, P30-5500) and 10% dimethyl sulfoxide (Sigma D2650). Tumor samples or pleural effusions were obtained from patients with cancer undergoing tumor resections at University Hospital Basel or Kantonsspital Baselland Liestal, Switzerland. Patient characteristics are summarized in online supplemental tables S1 and S2 . Tumor samples were mechanically dissociated and digested into single cell suspensions using accutase (Innovative Cell Technologies, AT-104), collagenase IV (Worthington, LS004188), hyaluronidase (Sigma-Aldrich, H6254) and DNAse type IV (Sigma-Aldrich, D5025). Single-cell suspensions were then stored in liquid nitrogen until further use. In the in vitro assays, single-cell suspensions derived from cancer samples were maintained in RPMI medium containing L-glutamine (Sigma-Aldrich, R8758) supplemented with 1x penicillin/streptomycin (Sigma-Aldrich, P4333), 1 mM pyruvate (Sigma-Aldrich), 1% non-essential amino acids (NEAA); Sigma-Aldrich) and 10% FBS (Pan Biotech, P30-5500).
P30 5500
Manufactured by PAN Biotech
The P30-5500 is a high-performance laboratory centrifuge capable of processing samples at speeds up to 30,000 rpm. It features a compact design and is suitable for a wide range of applications requiring rapid and efficient sample separation.
Automatically generated - may contain errors
Lab products found in correlation
Pyruvate, by Merck Group (2 mentions)
Dnase type 4, by Merck Group (2 mentions)
Histopaque, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Collagenase 4, by Worthington (2 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
3 protocols using p30 5500
1
Isolation and Cryopreservation of Primary Cells
2
In Vitro Tumor Sample Treatment
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To set up the in vitro treatment of patient samples, 300,000 single cells from the tumor suspensions, prepared as described above, were seeded in 90 µL of RPMI medium containing L-glutamine (Sigma-Aldrich, R8758) supplemented with 1x penicillin/streptomycin (Sigma-Aldrich, P4333), 1 mM pyruvate (Sigma-Aldrich), 1% NEAA (Sigma-Aldrich) and 10% FBS (Pan Biotech, P30-5500) in a 96-well flat bottom plate; 10 µL of 10× concentrated Abs (optimal dose calculated based on unpublished observations), PD1-TIM3 (RO7121661, RG7768, Roche), PD1-LAG3 (RO7247669, RG6139, Roche), anti-PD-1 (pembrolizumab, human IgG1 containing the P329G-LALA mutation, Roche) or DP47 isotype control (Roche) were then added to the cultures to obtain a final concentration of 10 µg/mL. The treated cells were incubated at 37°C, 5% CO2 for 48 or 96 hours before flow cytometry, cytokine analysis or scRNAseq preparation.
3
Isolation and Culture of Ovarian Cancer Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation, using Histopaque (Sigma-Aldrich, 10771), from buffy coats obtained from healthy blood donors (Blood Bank, University Hospital Basel, Switzerland). Fresh tumor samples were obtained from two ovarian cancer patients undergoing tumor resections at University Hospital Basel, Switzerland. Patient characteristics are summarized in Supplementary Table 1 . The study was approved by the local Ethical Review Board (Ethikkommission Nordwestschweiz) and University Hospital Basel, Switzerland. Written consent to use their tumor samples for research purposes was obtained from all patients. Fresh tumor samples were mechanically dissociated and digested using accutase (Innovative Cell Technologies, AT-104), collagenase IV (Worthington, LS004188), hyaluronidase (Sigma-Aldrich, H6254), and DNAse type IV (Sigma-Aldrich, D5025), directly after excision. Single-cell suspensions were prepared and samples were stored in liquid nitrogen until further use. In the following assays, single-cell suspensions derived from ovarian cancer samples were maintained in RPMI medium containing L-glutamine (Sigma-Aldrich, R8758) supplemented with 1 × penicillin/streptomycin (Sigma-Aldrich, P4333) and 10% FBS (Pan Biotech, P30-5500).
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