Ckx41sf microscope

10³ KU812 cells were seeded in methylcellulose without supplement of cytokines (MethoCult, 03231; STEMCELL Technologies, Vancouver, BC, Canada). Colonies were counted after 14 days and photographed using a CKX41SF microscope and a DP21 digital camera (both from Olympus). Single colonies were picked for quantitative real‐time polymerase chain reaction (qPCR).

The growth of A. protothecoides was measured by the number of cells monitored using the Bürker chamber under an OLYMPUS/CKX41SF microscope. The specific growth rate was calculated in the exponential phase using the equation:
where µ is the specific growth rate, and N₁ and N₂ are the numbers of cells at the start (t₁) and the end of the exponential growth phase (t₂).

EA.hy926 cells were seeded at 5 × 10⁴ cells per well coated with 200 μL/well of Matrigel (Becton Dickinson Labware, Bedford, MA, USA) and incubated for 4 h at 37°C in DMEM medium with or without calf thymus histones (Roche Diagnostics). The capillary-like structures were then examined under an Olympus CKX41SF microscope (Olympus Co., Tokyo, Japan), and tubule length was calculated in four-randomly selected fields using Image J software (NIH, Bethesda, MD, USA).

The Matrigel reagent (BD Biosciences) was used for the angiogenesis assay HMEC1/ECM-GFR (ECM (extracellular matrix); GFR (growth factor reduced)). This was handled according to the manufacturer's instructions, using precooled tubes and tips to prevent premature gelling. The angiogenesis assays were performed in Ibidi μ-slides (15 wells per slide). Briefly, the cell culture HMEC1, which had a confluence of 80%, was removed from the culture medium and MCDB131 medium with 2% SFB and antibiotic was added. Cells were incubated for 24 hrs. The next day, 10 μL of Matrigel per well (μ-slide plate) was added at a concentration of 8.2 mg/mL. The plate was incubated for 30 to 60 minutes at 37°C to achieve jellification. Then, 50 μL of HMEC1 cells was added at a concentration of 4 × 10⁵ cells/mL and the plate was incubated again at 37°C for 1 hour. After this time, 10 μL of cell supernatants was added under various conditions. The μ-slides were incubated for 24 hrs at 37°C in a humid atmosphere containing 5% CO₂. After the incubation time, μ-slides were photographed with the 590CU 5.0M CCD camera coupled to the OLYMPUS CKX41SF microscope.

Site-saturated mutagenesis was performed by overlapping PCR with degenerate primers (Table 5). PCRs were carried out sequentially with each pair of primers using Pfu DNA polymerase. DNA with random substitutions in two positions (105 and 117) was cloned into the pEt22b vector and transformed into E. coli BL21(DE3) cells. The resulting colonies were transferred to LB agar medium with IPTG and grown at 20 °C for 24 h. Colonies were analyzed for fluorescence on an Olympus CKX41SF microscope by irradiation with excitation light at 470 nm.Table 5

Degenerate primers for overlapped PCR.

Primer	Sequence
C105X_fw	GAT GGC GGA TTT NNN ACA GTC AGT GCA
C105X_rev	TGC ACT GAC TGT NNN AAA TCC GCC ATC
C117X_fw	GAC AAC NNN TTC ATT CAC ACA TCC ATG
C117X_rev	TGT GTG AAT GAA NNN GTT GTC TTT AAG TTT TAT