P. murina and P. carinii were partially purified by Ficoll-Hypaque density gradient centrifugation (49 (link)) and frozen at −80°C in cell recovery media (GIBCO). For short-term Pneumocystis cultures, A549 (ATCC) and LET1 (a gift from Dr. Paul Thomas, St. Jude Children’s Research Hospital) cells were cultured in culture medium F12 with 2.5% or 5% heat inactivated fetal bovine serum (GIBCO) and Penicillin-Streptomycin (GIBCO), plated to approximately 60%–80% confluency and incubated for 24 hours at 37°C. The next day, frozen Pneumocystis vials were thawed, washed in 50 mL 1× PBS, and centrifuged at 2,000 g for 20 minutes. Pneumocystis cell pellets were resuspended in culture medium and added to the plated cells. Media were partially changed every 3 or 4 days. At the set time points (days 7 and 14), wells were scraped for collection. Cell/organism suspensions were centrifuged at 10,000 g for 3 min, the supernatant was removed, and the pellets were frozen at −80°C. Genomic DNA was extracted using the QiAmp DNA Extraction Kit (Qiagen). Quantitative PCR targeting the single copy dhfr gene was performed as described previously (50 (link)).
Cell recovery media
Manufactured by Thermo Fisher Scientific
Cell recovery media is a laboratory product designed to support the viability and recovery of cells during various cell culture and handling procedures. It provides a balanced solution to maintain the optimal conditions for cells, promoting their survival and enabling efficient cell recovery.
Automatically generated - may contain errors
2 protocols using cell recovery media
1
Pneumocystis Long-Term Culturing Protocol
2
Pneumocystis Cultivation and Quantification
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P. murina and P. carinii were partially purified by Ficoll-Hypaque density gradient centrifugation (Kovacs, Halpern et al. 1988 (link)) and frozen at −80°C in cell recovery media (Gibco). For short term Pneumocystis cultures, A549 (ATCC) and LET1 (a gift from Dr. Paul Thomas, St Jude Children’s Research Hospital) cells were cultured in culture medium F12 media with 2.5% or 5% heat inactivated fetal bovine serum (GIBCO) and Penicillin-Streptomycin (GIBCO), plated to approximately 60 – 80% confluency and incubated for 24h at 37°C. The next day, frozen Pneumocystis vials were thawed, washed in 50ml 1x PBS, and centrifuged at 2,000g for 20 minutes. Pneumocystis cell pellets were resuspended in culture medium and added to the plated cells. Media were partially changed every 3 or 4 days. At set time points, wells were scraped for collection. Cell/organism suspensions were centrifuged at 10,000g for 3 min, the supernatant was removed, and the pellets were frozen at −80°C. Genomic DNA was extracted using QiAmp DNA Extraction Kit (Qiagen). Quantitative PCR targeting the single copy dhfr gene was performed as described previously (Liu, Davis et al. 2020 (link)).
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